WO2025091008A1

WO2025091008A1 - Three-dimensional engineered lymphatic and associated tissue grafts

Info

Publication number: WO2025091008A1
Application number: PCT/US2024/053212
Authority: WO
Inventors: Omid Veiseh; Martha FOWLER; Shalini Pandey; Alvaro Moreno LOZANO; Julian Krause; Qixu ZHANG; Yewen WU
Original assignee: William Marsh Rice University; University of Texas System; University of Texas at Austin
Current assignee: William Marsh Rice University; University of Texas System; University of Texas at Austin
Priority date: 2023-10-27
Filing date: 2024-10-28
Publication date: 2025-05-01
Anticipated expiration: 2026-04-27

Abstract

Described herein are methods of use involving tissue mimicking compositions comprising hydrogel systems and at least one therapeutic agent

Description

THREE-DIMENSIONAL ENGINEERED LYMPHATIC AND ASSOCIATED TISSUE

GRAFTS

[0000] This application is an International Application which claims priority from U.S. provisional patent application no. 63/593,840, filed on October 27, 2023, U.S. provisional patent application no. 63/635,145, filed on April 17, 2024, and U.S. provisional patent application 63/668,540, filed on July 08, 2024, the entire contents of each of which are incorporated herein by reference.

[0001] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

[0002] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.

BACKGROUND OF THE INVENTION

[0003] Field of the Invention

[0004] The present disclosure relates to the fields of biology, medicine, bioengineering and medical devices. More particularly, it relates to methods involving tissue compositions, such as engineered lymphatic vessel compositions or engineered blood vessel compositions. In particular, it relates to methods for use of implantable compositions designed to deliver therapeutic agents.

[0005] Description of Related Art

[0006] Encapsulation within semi-permeable hydrogels represents a local immuno-i solation strategy for many therapies without the need for systemic immunosuppression (Chang, 1964; Lim and Sun, 1980). The hydrogel sphere may facilitate the diffusion of nutrients necessary for cell function while excluding immune cells that would reject the foreign cells. Alginate spheres are one of the most widely investigated cell encapsulation materials because this anionic polysaccharide forms a hydrogel in the presence of divalent cations under cell friendly conditions. This natural co-polymer can then exhibit differential physical properties depending on the ratio and sequential arrangement of mannuronic and guluronic acid residues, the molecular weight, the concentration and the divalent cations used to form the gels (Strand et ah, 2017). For instance, alginate with a high guluronic-block content has a higher binding affinity for barium ions and will form a tighter, more stable network compared with the same alginate type crosslinked with calcium (Haug and Smidsrod, 1970). Additionally, electrostatic complexation of a positively charged polymer to the negatively charged alginate surface can provide an outer layer to reduce sphere porosity and increase sphere stability (Kollmer et al., 2015). A final outer alginate layer or chemical modification of the polycation used for coating has been investigated to reduce the positive surface charge density of the sphere (Kollmer et al., 2015; Mooranian et al., 2016; Kleinberger et al., 2016). Many alginate sphere formulations are produced with variations in the alginate concentration, the crosslinking ion or the inclusion or exclusion of a polycation layer.

[0007] However, ionically crosslinked alginate hydrogels have suffered from some major drawbacks that were detrimental to their ensuing biomedical application. For example, gelation of alginate with divalent cations is a poorly controlled interaction that could yield hydrogels with an inhomogeneous pore size distribution. Moreover, these hydrogels have limited long-term stability in physiological conditions due to the release of divalent cations into the surrounding media. Thus, there exists a need for methods for treatment and prevention of disease involving embedded alginate hydrogels.

SUMMARY OF THE INVENTION

[0008] The present disclosure provides methods for treatment or prevention of a disease or disorder involving the use of engineered tissue mimicking compositions. In some embodiments, the compositions comprise a therapeutic agent. Methods of making such compositions are also disclosed herein. The inventors have applied the latest in 3D bioprinting technology to form tissue mimicking compositions with long-term viability and functionality, and to permit engraftment of higher densities of therapeutic cells. In light of the requirements for effective functioning as tissue, the design criteria in some embodiments include, but are not limited to, a plurality of branched vessel mimics ranging in size from tens to hundreds of micrometers in diameter and substantially smooth inner walls that may minimize frictional drag and turbulence during fluid flow. In some embodiments, the vessel mimics are formed from semi-permeable biomaterial that facilitates contact between cells and therapeutic agents. In some embodiments, the present methods facilitate treatment or prevention of a disease or disorder associated with the lymphatic system, such as lymphedema associated with mastectomy. [0009] In one aspect, the present disclosure provides methods for treating a disease or disorder in a patient in need thereof, said method comprising embedding a tissue mimicking composition in the patient, wherein said tissue mimicking composition comprises:

(i) a hydrogel system, wherein said system comprises at least one of A, B or C, wherein:

A is a photo-responsive alginate, wherein the molecular weight is less than 95 kDa and wherein the methacrylation efficiency is from about 15% to about 95%;

B is a photo-responsive alginate, wherein the molecular weight is from about 55 kDa to about 240 kDa and wherein the methacrylation efficiency is from about 1% to about 65%; and

C is a photo-responsive alginate, wherein the molecular weight is from about 180 kDa to about 320 kDa and wherein the methacrylation efficiency from about 1% to about 25%; and

(ii) at least one therapeutic agent.

[0010] In some embodiments, the hydrogel system comprises a monomer of the formula:

wherein n and m are each independently at least 1.

[0011] In some embodiments, the tissue mimicking composition comprises at least one channel or lumen. In some embodiments, the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells. In some embodiments, the tissue mimicking composition comprises at least one channel suitable for perfusion with blood cells.

[0012] In some embodiments, the tissue mimicking composition further comprises polyethylene glycol diacrylate, gelatin methacrylate, a triazole-containing alginate, or any combination thereof. In some embodiments, the tissue mimicking composition comprises gelatin methacrylate. In some embodiments, the tissue mimicking composition comprises a triazole-containing alginate.

[0013] In some embodiments, at least one therapeutic agent is an engineered cell or a nonengineered cell. In some embodiments, at least one therapeutic agent is an engineered protein, a secreted protein, a hormone, a cytokine, an antibody, an enzyme, or a peptide. In some embodiments, at least one therapeutic agent is a small molecule. In some embodiments, at least one therapeutic agent is an agent used to treat or prevent inflammation.

[0014] In some embodiments, the tissue mimicking composition comprises at least two channels. In some embodiments, a boundary between adjacent channels of the tissue mimicking composition has one or more pores allowing contact between adjacent channels.

[0015] In some embodiments, the tissue mimicking composition resembles a lymph node.

[0016] In some embodiments, the disease or disorder is lymphedema or another lymphatic disease or disorder, an autoimmune disease or disorder, a metabolic disease or disorder, cancer, a neurological disease or disorder, inflammation, or cardiovascular disease. In some embodiments, the disease or disorder is lymphedema or another lymphatic disease or disorder. In some embodiments, the disease or disorder is lymphedema.

[0017] In another aspect, the present disclosure provides methods of making a tissue mimicking composition as described above comprising: a) obtaining at least one ink composition; and b) depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology; to form a tissue mimicking composition.

[0018] In some embodiments, the method further comprises exposing the deposited ink pattern to light.

[0019] Any embodiment of any of the present devices, methods, compositions, kits, and systems may consist of or consist essentially of, rather than comprise/include/contain/have, the described steps and/or features. Thus, in any of the claims, the term “consisting of’ or “consisting essentially of ’ may be substituted for any of the open-ended linking verbs recited above, in order to change the scope of a given claim from what it would otherwise be using the open-ended linking verb.

[0020] Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0022] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0023] FIG. 1 shows schematic diagrams of exemplary single (left) and dual (right) tissue mimicking compositions. LV: lymphatic vessel; BV: blood vessel.

[0024] FIG. 2 illustrates an exemplary tissue mimicking composition comprising interconnected vessel mimicking channels. LV: lymphatic vessel; BV: blood vessel.

[0025] FIG. 3 illustrates an exemplary tissue mimicking composition according to the present disclosure. The arrangement of vessel mimicking channels and semipermeable biomaterial shown here resembles a lymph node. Accordingly, the present disclosure provides tissue mimicking compositions that resemble or function as an engineered lymph node.

[0026] FIG. 4 provides, at top, 3D renderings of open channel architecture software designs prior to 3D bioprinting. Bottom: 3D printed hydrogels with open and perfusable channels. Whole hydrogel dimensions are between 16mm length and 8mm wide with channel dimensions between 0.5-lmm.

[0027] FIG. 5 illustrates epifluorescence microscopy images of lymphatic endothelial cells (LEC) perfusion within 3D printed hydrogel, scale bar = 100 um. [0028] FIG. 6 shows confocal microscopy images of 3D printed channel section for lymphatic endothelial cells (LEC) and nuclei within the channel after 6 days in perfusion culture, scale bar = 100 um.

[0029] FIG. 7 illustrates various types of cell engineered platforms described herein including non-encapsulated clusters (panel A), micro-encapsulated engineered cells (panel B), macroencapsulated engineered cells (panel C), and perfused engineered cells (panel D).

[0030] FIG. 8 shows schematic of engineering method to secrete pro-angiogenic and pro- lymphogenic growth factors.

[0031] FIG. 9 depicts graphs showing secretion of 6 pro-angiogenic growth factors using the cell engineering platform described herein. From left to right FGF-21, FGF-2, PDGF-A, EGF, VEGF- A and Pl GF.

[0032] FIG. 10 depicts graphs showing secretion of pro-lymphogenic growth factor VEGF-C that is secreted using the cell engineering platform.

[0033] FIG. 11 shows secretion of PDGF-A and EGF by pro-angiogenic cells encapsulated in 0.4 mm small molecule modified sodium alginate microcapsules and implanted in the subQ space of SKH1 ELITE mice. After injection of integrisense a neovascularization probe, IVIS imaging shows increased pro-angiogenic signal compared to un engineered cells.

[0034] FIG. 12 shows gross images (top row) and histology pictures (bottom row) of mouse tissue surrounding implanted alginate capsules with PDGF-A and EGF engineered secreting cells after 7 days in vivo.

[0035] FIG. 13 shows local and systemic levels of EGF after implanting small molecule modified 0.4 mm sodium alginate capsules in the subQ space of SKH1 ELITE mice for 1 day.

[0036] FIG. 14 shows secretion of FGF-21 and VEGF-A by pro-angiogenic cells encapsulated in 0.4 mm small molecule modified sodium alginate microcapsules and implanted in the subQ space of SKH1 ELITE mice. After injection of integrisense a neovascularization probe, IVIS imaging shows increased pro-angiogenic signal compared to un engineered cells.

[0037] FIG. 15 shows histology pictures of mouse tissue surrounding implanted alginate capsules with FGF-21 and VEGF-A engineered secreting cells after 7 days in vivo.

[0038] FIG. 16 shows secretion of FGF-21 by pro-angiogenic cells encapsulated in 0.4 mm small molecule modified sodium alginate microcapsules and implanted in the subQ space of SKH1 ELITE mice. After injection of integri sense a neovascularization probe, IVIS imaging shows increased pro-angiogenic signal compared to un engineered cells.

[0039] FIG. 17 shows histology pictures of mouse tissue surrounding implanted alginate capsules with FGF-21 engineered secreting cells after 7 days in vivo.

[0040] FIG. 18 shows an implantable hydrogel platform engineered to guide and generate new lymphatic and blood vessels to the area of swelling to resolve chronic lymphedema.

[0041] FIG. 19 shows an implantable hydrogel platform engineered to produce VEGF-C to guide and generate new lymphatic and blood vessels to the area of swelling to resolve chronic lymphedema.

[0042] FIG. 20 shows MRI images and swelling measurements from lymph nodes extracted from a lymphedema mouse model (hindlimb) after radiation therapy and treatment with the platform composition described in FIG. 19.

[0043] FIG. 21 Programmable RPE-VEGF-C cell therapy enables stable and local production with inducible safety switch for termination, a, Overall schematic of workflow for engineering and characterization of engineered RPE-VEGFC cells with inducible small molecule for cell death (created withBioRender.com). b, VEGF-C production of VEGF-C engineered (RPE-VEGFC) and non-engineered (RPE), and ADSC cell lines over 24 hours in culture, c, VEGF-C production over time in RPE-VEGFC cell lines in culture, d, lymphatic tube formation assay of LECs with RPE- VEGFC and ADSC supplementation and LECs alone after one day in culture, e, RPE-VEGFC viability and production with engineered kill switch at 10 pM, I pM, lOOnM, lOnM, InM, O. lnM, O.OlnM, and OnM of small molecule activator

[0044] FIG. 22 Development of A1MA library and high-throughput screen to enable leading materials for RPE-VEGFC cell therapy, a, synthesis schematic of alginate methacrylate (A1MA) for development of A1MA formulation libraries, b, screening strategy for A1MA library, c, NMR and elemental analysis of methacrylateted alginate, d, gelation assay with Rhodamine B loaded A1MA formulation samples, e, shape retention of ionically crosslinked alginate with barium chloride, and A1MA at different methacrylation efficiencies upon UV crosslinking, f, mechanical strength of A1MA library formulations, g, in vitro stability of A1MA formulations after 28 days under cell culture conditions, h, diffusion of VEGF-C after 24 hours from encapsulated RPE- VEGFC cells in A1MA formulations that were mechanically strong and stable. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0045] Detailed descriptions of one or more preferred embodiments are provided herein. It is to be understood, however, that the present invention may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner.

[0046] The singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0047] Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly “an example,” “exemplary” and the like are understood to be nonlimiting.

[0048] The term “substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited.

[0049] The terms “comprising” and “including” and “having” and “involving” (and similarly “comprises”, “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a, b, and c” means that the process includes at least steps a, b and c. Wherever the terms “a” or “an” are used, “one or more” is understood, unless such interpretation is nonsensical in context.

[0050] The term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).

[0051] As used herein, the terms “drug”, “pharmaceutical”, “therapeutic agent”, and “therapeutically active agent” are used interchangeably to represent a compound which invokes a therapeutic or pharmacological effect in a human or animal and is used to treat a disease, disorder, or other condition. In some embodiments, these compounds have undergone and received regulatory approval for administration to a living creature.

[0052] The term “effective,” as that term is used in the specification and/or claims, means adequate to accomplish a desired, expected, or intended result. “Effective amount,” “Therapeutically effective amount” or “pharmaceutically effective amount” when used in the context of treating a patient or subject with a compound means that amount of the compound which, when administered to the patient or subject, is sufficient to effect such treatment or prevention of the disease as those terms are defined below.

[0053] As used herein, the term “IC50” refers to an inhibitory dose which is 50% of the maximum response obtained. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological, biochemical or chemical process (or component of a process, i.e. an enzyme, cell, cell receptor or microorganism) by half.

[0054] As used herein, the term “resembles” (and any form of “resembles” such as "resembling”) describes a likeness to a particular mentioned antecedent or inspiration. The likeness may be designed, a natural outcome of a design, or inherent. The likeness may be discernable to the artisan, such as by sight, by sound, by touch, by taste, or by smell. The likeness may be due to shared features between the inspiration and that which has a likeness to the inspiration. Not all of the features or properties of the inspiration to have a likeness to the inspiration. The likeness may be measurable. For example, a tissue mimicking composition that resembles lymphatic tissue or a lymph node may provide the same values or results as lymphatic tissue or a lymph node in one or more assays or according to one or more tests.

[0055] As used herein, the term “patient” or “subject” refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human patients are adults, juveniles, infants and fetuses.

[0056] As generally used herein “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio. [0057] “Pharmaceutically acceptable salts” means salts of compounds disclosed herein which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as 1,2 ethanedisulfonic acid, 2 hydroxyethanesulfonic acid, 2 naphthalenesulfonic acid, 3 phenylpropionic acid, 4,4' methylenebis(3 hydroxy 2 ene-1 carboxylic acid), 4 methylbicyclo[2.2.2]oct 2 ene-1 carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxy naphthoic acid, lactic acid, laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o (4 hydroxybenzoyl)benzoic acid, oxalic acid, p chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiarybutyl acetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N methylglucamine and the like. It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0058] ‘ ‘Prevention” or “preventing” includes: (1) inhibiting the onset of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease, and/or (2) slowing the onset of the pathology or symptomatology of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease. [0059] “Subject,” as used herein, refers to the recipient of the implantable composition described herein. The subject may include a human and/or other non-human animals, for example, mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys); commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and/or dogs) and birds (e.g., commercially relevant birds such as chickens, ducks, geese, and/or turkeys). In certain embodiments, the animal is a mammal. The animal may be a male or female and at any stage of development (e.g., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult). A non-human animal may be a transgenic animal.

[0060] Treatment” or “treating” includes (1) inhibiting a disease in a subject or patient experiencing or displaying the pathology or symptomatology of the disease (e.g., arresting further development of the pathology and/or symptomatology), (2) ameliorating a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease (e.g., reversing the pathology and/or symptomatology), and/or (3) effecting any measurable decrease in a disease or symptom thereof in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease. In some embodiments, “treatment,” “treat,” and “treating” require that signs or symptoms of the disease or condition have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease or condition, e.g., in preventive treatment. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence. Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.

[0061] As used in this specification, the term “significant” (and any form of significant such as “significantly”) is not meant to imply statistical differences between two values but only to imply importance or the scope of difference of the parameter.

[0062] As used herein, the term “substantially free of’ or “substantially free” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of all containments, by-products, and other material is present in that composition in an amount less than 2%. The term “more substantially free of’ or “more substantially free” is used to represent that the composition contains less than 1% of the specific component. The term “essentially free of’ or “essentially free” contains less than 0.5% of the specific component.

[0063] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements and parameters.

[0064] The above definitions supersede any conflicting definition in any reference that is incorporated by reference herein. The fact that certain terms are defined, however, should not be considered as indicative that any term that is undefined is indefinite. Rather, all terms used are believed to describe the invention in terms such that one of ordinary skill can appreciate the scope and practice the present invention.

[0065] Provided herein are methods of use of tissue mimicking compositions and methods of making such compositions. The compositions of the presently disclosed methods have the advantage of a modular design. In some embodiments, the compositions of the presently disclosed methods are tunable, such as by altering the identity of any therapeutic agents which form a part of the compositions. In some embodiments, the methacrylation efficiency of the polymer used in the present methods may result in advantageous embodiments of the present disclosure. In some embodiments, the presence of structural features and/or the shape thereof in hydrogels used in the present disclosure may result in advantageous embodiments of the present disclosure. In some embodiments, the molecular weight of the hydrogels used in the present disclosure may result in advantageous embodiments of the present disclosure. In some embodiments, the tissue mimicking compositions used herein are three-dimensional. In some embodiments, the compositions of the presently disclosed methods comprise one type of tissue mimic. In other embodiments, the compositions of the presently disclosed methods may comprise multiple tissue mimics. In some embodiments, the tissue mimicking composition of the methods disclosed herein resembles a lymph node. In some embodiments, adjacent tissue mimics may be separated by semipermeable biomaterial. In other embodiments, adjacent tissue mimics may be interconnected, or the barrier between adjacent tissue mimics may be perforated to allow transfer of material between adjacent tissue mimicking regions. In some embodiments, the compositions of the presently disclosed methods may have a variety of channel diameters, cellular densities, or flowrates in order to serve as a tissue mimic. In some embodiments, the compositions of the presently disclosed methods may be formed using light-based bioprinting or casting techniques. In some embodiments, the compositions of the presently disclosed methods are formed from a hydrogel. In some embodiments, the hydrogel is photo-responsive. These aspects and more are described below and in the sections that follow.

[0066] I. Hydrogel Compositions

[0067] The presently disclosed methods involve the use of tissue mimicking compositions, wherein said compositions comprise a hydrogel polymer. The hydrogel compositions usable in the present methods that are stable, permeable, perfusable, or biocompatible, or any other advantage identifiable by the artisan. In some embodiments, the presently disclosed methods involve release of encapsulated therapeutic agents or perfusion of therapeutic agents across a hydrogel. In some embodiments, the hydrogel used in the presently disclosed methods may be altered or optimized to improve any advantages of the invention or to obtain a particular property desirable to the artisan. In some embodiments, the formulation of the hydrogel used in the presently disclosed methods may be selected to achieve a certain level of stability, permeability, perfusability, or biocompatibility. In some embodiments, the method involves use of a hydrogel possessing certain features, such as for example perforations, channels, or lumens, to facilitate or improve vascularization or any of the above-mentioned aspects. In some embodiments, the formulation of the hydrogel used in the presently disclosed methods may be selected to facilitate a certain rate of biodegradation for the platform. In some embodiments, the size of the hydrogel used in the presently disclosed methods may be selected to facilitate a certain rate of biodegradation for the platform.

[0068] One embodiment provides for the use of modified alginate polymers in therapeutic agentcontaining implants. Modified alginate polymers can be of any desired molecular weight. The weight average molecular weight of the alginates is preferably between 1,000 and 1,000,000 Daltons, more preferably between 10,000 and 500,000 Daltons as determined by gel permeation chromatography. Unmodified alginate typically has a weight average molecular weight of about 50,000 Daltons to about 500,000 Daltons; however, unmodified alginates having molecular weights outside this range can also be used. In some embodiments, the average molecular weight is less than 95,000 Daltons, preferably less than 75,000 Daltons. In some embodiments, the average molecular weight is from about 50,000 to about 250,000 Daltons, preferably from about 75,000 Daltons to about 220,000 Daltons. In some embodiments, the average molecular weight is from about 175,000 to about 325,000 Daltons, preferably from about 200,000 Daltons to about 300,000 Daltons.

[0069] Modified alginate polymers can contain any ratio of covalently modified monomers. In some embodiments, greater than 2.5%, 5%, 7.5%, 10%, 12%, 14%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 26%, 28%, 30%, 32.5%, 35%, 37.5%, 40%, 45%, 50%, 55%, or 60% of the monomers in the modified alginate polymer are covalently modified monomers. Greater than 10%, greater than 20%, or greater than 30% of the monomers in the modified alginate polymer are covalently modified monomers.

[0070] Modified alginate polymers can be produced incorporating covalently modified monomers possessing a range of different hydrogen bonding potentials, hydrophobicities/hydrophilicities, and charge states. The inclusion of covalently modified monomers into an alginate polymer alters the physiochemical properties of alginate polymer. Accordingly, the physiochemical properties of alginates can be tuned for desired applications by the selective incorporation of covalently modified monomers.

[0071] For example, the glass transition temperature (T_g), can be varied by the incorporation of covalently modified monomers. In some embodiments, the modified alginate polymer powder possess a T_g, as measured by differential scanning calorimetry (DSC), of greater than 50°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C, 105°C, 110°C, 115°C, 120°C, 125°C, 130°C, 135°C, 140°C, 145°C, 150°C, 160°C, 175°C, 190°C, or 200°C.

[0072] The hydrophobicity/hydrophilicity of alginates can be varied by the incorporation of hydrophobic and/or hydrophilic covalently modified monomers. In particular embodiments, the modified alginate polymer contains one or more hydrophobic covalently modified monomers. The relative hydrophobicity/hydrophilicity of modified alginates can be quantitatively assessed by measuring the contact angle of a water droplet on a film of the modified alginate polymer using a goniometer. In some embodiments, the modified alginate has a contact angle of less than 90° (i.e. it is hydrophilic). In particular embodiments, the modified alginate has a contact angle of more than 90° (i.e., it is hydrophobic). In some embodiments, the modified alginate has a contact angle of more than 95°, 100°, 105°, 110°, 115°, or 120°. [0073] In embodiments used for cell encapsulation, the modified alginate polymer can be ionically crosslinked by a polyvalent cation such as Ca²⁺, Sr²⁺, or Ba²⁺ to form hydrogels.

[0074] In some embodiments, the modified alginate polymer forms hydrogels such that the fluorescence intensity measured using the high throughput hydrogel formation assay described herein is greater than 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, or 55,000. In particular embodiments, the modified alginate polymer forms hydrogels such that the fluorescence intensity measured using the high throughput hydrogel formation assay described herein is greater than 15,000. In particular embodiments, the modified alginate polymer forms hydrogels such that the fluorescence intensity measured using the high throughput hydrogel formation assay described herein is between 15,000 and 55,000, preferably between 20,000 and 55,000, more preferably between 25,000 and 55,000.

[0075] The porosity and surface area of modified alginates can be measured using BET analysis. Prior to BET analysis, solvent and volatile impurities are removed by prolonged heating of the modified alginate gel under vacuum. Subsequently, the hydrogel samples are cooled under vacuum, for example by liquid nitrogen, and analyzed by measuring the volume of gas (typically N2, Kr, CO2, or Ar gas) adsorbed to the hydrogel at specific pressures. Analysis of the physisorption of the gas at variable pressures is used to characterize the total surface area and porosity of gels formed by the modified alginate polymers. A particular method of determining hydrogel porosity is BET analysis.

[0076] In particular embodiments, the modified alginate forms a hydrogel with sufficient porosity to permit nutrients, waste, and the hormones and/or proteins secreted from encapsulated cells to diffuse freely into and out of the capsules, while simultaneously preventing the incursion of immune cells into the gel matrix. In some embodiments, the porosity of the hydrogel formed by the modified alginate polymer is increased by 5%, 10%, 15%, or 20% relative to the porosity of a hydrogel formed from the unmodified alginate polymer. In alternative embodiments, the porosity of the hydrogel formed by the modified alginate polymer is decreased by 5%, 10%, 15%, or 20% relative to the porosity of a hydrogel formed from the unmodified alginate polymer.

[0077] In particular embodiments used for cell encapsulation, the modified alginate is biocompatible. The biocompatibility of modified alginates can be quantitatively determined using a fluorescence-based in vivo biocompatibility assay. [0078] In some embodiments, the modified alginate polymer is biocompatible such that the fluorescence response normalized to unmodified alginate measured using the in vivo biocompatibility assay described herein is less than 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%. In particular embodiments, the modified alginate polymer induces a lower foreign body response than unmodified alginate. This is indicated by fluorescence response normalized to unmodified alginate of less than 100%. In some embodiments, the modified alginate polymer is biocompatible such that the fluorescence response normalized to unmodified alginate measured using the in vivo biocompatibility assay described herein is less than 75%, more preferably less than 65%, and most preferably less than 50%.

[0079] The modified alginates can be chemically modified as described herein to any desired density of modifications. The density of modifications is the average number of modifications (that is, attached compounds) per a given weight, volume, or area of the surface of a capsule or product that includes the modified alginate. Generally, a density at or above a threshold density can provide a beneficial effect, such as lower foreign body response. In some embodiments, a high density is not required. Without being bound to any particular theory of operation, it is believed that the chemical modifications signal to, indicate to, or are identified by, one or more immune system or other body components to result in a beneficial effect, such as a lower foreign body response. In some embodiments, a lower density of modifications can be effective for this purpose.

[0080] Useful densities include densities of at least, of less than, of about, or of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, and 1000 modifications per square piq, per pg, or per cubic pqi. All ranges defined by any pair of these densities are also specifically contemplated and disclosed.

[0081] In some embodiments, the density of the modifications on a surface, surfaces, or portions of a surface(s) of a capsule or product that, when the product is administered to (e.g., implanted in the body of) a subject, would be in contact with fluid(s), cell(s), tissue(s), other component(s), or a combination thereof of the subject's body is greater than the density of the modifications on other surfaces of the product.

[0082] Density can also be expressed in terms of the concentration of the surface modifications as measured by X-ray photoelectron spectroscopy (XPS). XPS is a surface-sensitive quantitative spectroscopic technique that measures the elemental composition at the parts per thousand range of the elements that exist within a material.

[0083] XPS spectra are obtained by irradiating a material with a beam of X-rays while simultaneously measuring the kinetic energy and number of electrons that escape from the top 0 to 10 nm of the material being analyzed. By measuring all elements present on the surface, the percentage of the elements that come from the surface modifications can be calculated. This can be accomplished by, for example, taking the percentage of nitrogen (and/or other elements in the surface modifications) in the total elemental signal measured. Nitrogen is a useful indicator for the surface modification because many substrated and materials forming the capsule or product contain little nitrogen. For convenience, the percent of the element(s) used to indicate the surface modifications can be stated as the percent surface modifications. Also for convenience, the percent surface modifications can be referred to as the concentration of surface modifications.

[0084] Useful percent surface modifications include concentrations of about, less than or at 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100 percent surface modifications. All ranges defined by any pair of these concentrations are also specifically contemplated and disclosed.

[0085] In one aspect, the present methods may involve alginate, including modified alginates, that have been used to form a capsule. Capsules are particles having a mean diameter of about 150 pm to about 5 cm. The disclosed capsules can be formed of cross-linked hydrogel. Other than the encapsulated material, the capsules, for example, can be formed solely of cross-linked hydrogel, can have a cross-linked hydrogel core that is surrounded by one or more polymeric shells, can have one or more cross-linked hydrogel layers, can have a cross- linked hydrogel coating, or a combination thereof. The capsule may have any shape suitable for, for example, cell encapsulation. The capsule may contain one or more cells dispersed in the cross-linked hydrogel, thereby "encapsulating" the cells. Particular capsules are formed of or include one or more of the disclosed modified alginates.

[0086] Capsules can have a mean diameter of about 150 pm to about 8 mm. Capsules can have any mean diameter from about 150 pm to about 5 cm. In particular, the capsules have a mean diameter that is greater than 1 mm, more particularly 1.5 mm or greater. In some embodiments, the capsules can be as large as about 8 mm in diameter. For example, the capsule can be in a size range of about 1 mm to 8 mm, 1 mm to 6 mm, 1 mm to 5 mm, 1 mm to 4 mm, 1 mm to 3 mm, 1 mm to 2 mm, 1 mm to 1.5 mm, 1.5 mm to 8 mm, 1.5 mm to 6 mm, 1.5 mm to 5 mm, 1.5 mm to 4 mm, 1.5 mm to 3 mm, or 1.5 mm to 2 mm.

[0087] The rate of molecules entering the capsule necessary for cell viability and the rate of therapeutic products and waste material exiting the capsule membrane can be selected by modulating capsule permeability. Capsule permeability can also be modified to limit entry of immune cells, antibodies, and cytokines into the capsule. Generally, as shown by the examples, known methods of forming hydrogel capsules can produce capsules the permeability of which limit entry of immune cells, antibodies, and cytokines into the capsule. Since different cell types have different metabolic requirements, the permeability of the membrane can be optimized based on the cell type encapsulated in the hydrogel. The diameter of the capsules is an important factor that influences both the immune response towards the cell capsules as well as the mass transport across the capsule membrane.

[0088] In other embodiments, one or more additional hydrogel-forming polymers are used in combination with unmodified alginate or in place of unmodified alginate. Such polymers are known in the art. Examples include, but are not limited to, PEG, chitosan, dextran, hyaluronic acid, silk, fibrin, poly(vinyl alcohol) and poly(hydroxyl ethyl methacrylate).

[0089] The particles prepared from a mixture of modified alginate and unmodified alginate produced more homogenous microparticle populations in terms of shape and size as evaluated by scanning electron microscopy (SEM).

[0090] In some embodiments, the hydrogel capsules can have any suitable shape. Useful shapes include spheres, sphere-like shapes, spheroids, spheroid-like shapes, ellipsoids, ellipsoid-like shapes, stadiumoids, stadiumoid-like shapes, disks, disk-like shapes, cylinders, cylinder-like shapes, rods, rod-like shapes, cubes, cube-like shapes, cuboids, cuboid-like shapes, toruses, toruslike shapes, and flat and curved surfaces. Products, devices, and surfaces that have been or will be coated can have any of these shapes or any shape suitable for the product or device.

[0091] Spheres, spheroids, and ellipsoids are shapes with curved surfaces that can be defined by rotation of circles, ellipses, or a combination around each of the three perpendicular axes, a, b, and c. For a sphere, the three axes are the same length. For oblate spheroids (also referred to as oblate ellipsoids of rotation), the length of the axes are a = b > c. For prolate spheroids (also referred to as prolate ellipsoids of rotation), the length of the axes are a = b < c. For tri-axial ellipsoids (also referred to as scalene ellipsoids), the length of the axes are a > b > c. Stadiumoids are rotational shapes of stadiums. Cylinders are rotational shapes of rectangles rotated on the long axis. Disks are squashed cylinders where the diameter is greater than the height. Rods are elongated cylinders where the long axis is ten or more times the diameter.

[0092] "Sphere-like shape," "spheroid-like shape," "ellipsoid-like shape," "stadiumoid- like shape," "cylinder-like shape," "rod-like shape," "cube-like shape," "cuboid-like shape," and "toruslike shape" refers to an object having a surface that roughly forms a sphere, spheroid, ellipsoid, stadiumoid, cylinder, rod, cube, cuboid, or torus, respectively. Beyond a perfect or classical form of the shape, a sphere-like shape, spheroid-like shape, ellipsoid-like shape, stadiumoid-like shape, cylinder-like shape, rod- like shape, cube-like shape, cuboid-like shape, and torus-like shape can have waves and undulations.

[0093] Generally, a sphere-like shape is an ellipsoid (for its averaged surface) with semi- principal axes within 10% of each other. The diameter of a sphere or sphere-like shape is the average diameter, such as the average of the semi-principal axes. Generally, a spheroid-like shape is an ellipsoid (for its averaged surface) with semi-principal axes within 100% of each other. The diameter of a spheroid or spheroid-like shape is the average diameter, such as the average of the semi-principal axes. Generally, an ellipsoid-like shape is an ellipsoid (for its averaged surface) with semi-principal axes within 100% of each other. The diameter of an ellipsoid or ellipsoid-like shape is the average diameter, such as the average of the semi-principal axes. Generally, a stadiumoid-like shape is a stadiumoid (for its averaged surface) with semi-principal axes of the ends within 20% of each other. The diameter of a stadiumoid or stadiumoid-like shape is the average diameter, such as the average of the semi-principal axes. Alternatively, the size of a stadiumoid or stadiumoid-like shape can be given as the average of the long axis. Generally, a cylinder-like shape is a cylinder (for its averaged surface) with semi- principal axes within 20% of each other. The diameter of a cylinder or cylinder-like shape is the average diameter, such as the average of the semi-principal axes.

[0094] Alternatively, the size of a cylinder or cylinder-like shape can be given as the average of the long axis. Generally, a rod-like shape is a rod (for its averaged surface) with semi- principal axes within 10% of each other. The diameter of a rod or rod-like shape is the average diameter, such as the average of the semi-principal axes. Alternatively, the size of a rod or rod-like shape can be given as the average of the long axis. Generally, a cubelike shape is a cube (for its averaged surface) with sides within 10% of each other. The diameter of a cube or cube-like shape is the average side length. Generally, a cuboid-like shape is a cuboid (for its averaged surface) with matching sides within 10% of each other. The diameter of a cuboid or cuboid-like shape is the average side length.

[0095] Generally, a torus-like shape is a torus (for its averaged surface) with semi-principal axes within 10% of each other. The diameter of a torus or torus-like shape is the average diameter, such as the average of the semi-principal axes. Alternatively, the size of a torus or torus-like shape can be given as the diameter across the ring.

[0096] "Flat side" refers to a contiguous area of more than 5% of a surface that has a curvature of 0. "Sharp angle" refers to a location on a surface across which the tangent to the surface changes by more than 10% over a distance of 2% or less of the circumference of the surface. Edges, comers, grooves, and ridges in a surface are all forms of sharp angles.

[0097] Particular capsules can be made of biocompatible materials, have a diameter of at least 1 mm and less than 10 mm, has a spheroid-like shape, and have one or more of the additional characteristics: surface pores of the capsules greater than 0 nm and less than 10 pm; surface of the capsules neutral or hydrophilic; curvature of the surface of the capsules at least 0.2 and is not greater than 2 on all points of the surface; and surface of the capsules lacking flat sides, sharp angles, grooves, or ridges. Generally, the capsules elicit less of a fibrotic reaction after implantation than the same capsules lacking one or more of these characteristics that are present on the capsules. [0098] In some embodiments, the capsules are provided as a preparation and at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the capsules in the preparation have a shape characteristic described herein, e.g., have a spheroid-like shape, or have a curvature of the surface of at least 0.2 to 2.0 on all points of the surface.

[0099] In some embodiments, the hydrogel capsules have a mean diameter that is greater than 1 mm, particularly 1.5 mm or greater. In some embodiments, the hydrogel capsules can be as large as 8 mm in diameter. For example, the hydrogel capsules is in a size range of 1 mm to 8 mm, 1 mm to 6 mm, 1 mm to 5 mm, 1 mm to 4 mm, 1 mm to 3 mm, 1 mm to 2 mm, 1 mm to 1.5 mm,

1.5 mm to 8 mm, 1.5 mm to 6 mm, 1.5 mm to 5 mm, 1.5 mm to 4 mm, 1.5 mm to 3 mm, 1.5 mm to 2 mm, 2 mm to 8 mm, 2 mm to 7 mm, 2 mm to 6 mm, 2 mm to 5 mm, 2 mm to 4 mm, 2 mm to 3 mm, 2.5 mm to 8 mm, 2.5 mm to 7 mm, 2.5 mm to 6 mm, 2.5 mm to 5 mm, 2.5 mm to 4 mm,

2.5 mm to 3 mm, 3 mm to 8 mm, 3 mm to 7 mm, 3 mm to 6 mm, 3 mm to 5 mm, 3 mm to 4 mm, 3.5 mm to 8 mm, 3.5 mm to 7 mm, 3.5 mm to 6 mm, 3.5 mm to 5 mm, 3.5 mm to 4 mm, 4 mm to 8 mm, 4 mm to 7 mm, 4 mm to 6 mm, 4 mm to 5 mm, 4.5 mm to 8 mm, 4.5 mm to 7 mm, 4.5 mm to 6 mm, 4.5 mm to 5 mm, 5 mm to 8 mm, 5 mm to 7 mm, 5 mm to 6 mm, 5.5 mm to 8 mm, 5.5 mm to 7 mm, 5.5 mm to 6 mm, 6 mm to 8 mm, 6 mm to 7 mm, 6.5 mm to 8 mm, 6.5 mm to 7 mm, 7 mm to 8 mm, or 7.5 mm to 8 mm. In some embodiments, the capsule has a mean diameter or size between 1 mm to 8 mm. In some embodiments, the capsule has a mean diameter or size between 1 mm to 4 mm. In some embodiments, the capsule has a mean diameter or size between 1 mm to 2 mm. In some embodiments, the capsules are provided as a preparation and at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the hydrogel capsules in the preparation have a diameter in a size range described herein.

[00100] In some embodiments, the disclosure provides methods involving use of hydrogels that have been formed from modified alginates. In some embodiments, the methods disclosed herein involve the use of a hydrogel that is photo-responsive, wherein the photo-responsive material comprises a monomer of Formula (I) represented by the following structural formula:

or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or a tautomer thereof, wherein n and m are each independently an integer from 1 to 100.

[0092] In some embodiments, the present methods involve the use of hydrogels that have been formed from very low viscosity alginates (VLV). Very low viscosity alginates are known in the art. An exemplary very low viscosity alginate is UPVLVG, which has a molecular weight less than 75 kDa. In some embodiments, the present methods involve the use of hydrogels that have been formed from very low viscosity alginates that have been modified, such as methacrylated. In some embodiments, the present methods involve the use of hydrogels that have been formed from low viscosity alginates (LV) that have been modified. Low viscosity alginates are known in the art. For example, SLG20 is a low viscosity alginate and has a molecular weight of 75-220 kDa. Another exemplary low viscosity alginate is SLG100, which has a molecular weight of 200-300 kDa. In some embodiments, the present methods involve the use of hydrogels that have been formed from very low viscosity alginates that have been modified, such as methacrylated. The present disclosure also provides for the use of hydrogels formed from any combination of modified or unmodified low viscosity or very low viscosity alginates. Formulations of hydrogels that are usable according to the current methods are described in further detail in the sections below and in Table 1 and Table 2.

[0093] In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 100%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, or about 25%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 95%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 90%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 85%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 80%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 75%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 70%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 65%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 60%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 55%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 50%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 45%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 40%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 35%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 30%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is less than about 25%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 20%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 46%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 60%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 90%. [0094] In embodiments, the methacrylation efficiency of the UPVLVG alginate is between about 10% and about 100%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is between about 12% and about 98%, about 14% and about 96%, about 16% and about 94%, about 18% and about 92%, or about 20% and about 90%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is between about 10% and about 100%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is between about 12% and about 98%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is between about 14% and about 96%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is between about 16% and about 94%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is between about 18% and about 92%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is between about 20% and about 90%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 20%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 46%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 60%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 90%. [0095] In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 5%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80% or about 85%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 5%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 10%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 15%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 20%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 25%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 30%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 35%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 40%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 45%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 50%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 55%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 60%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 65%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 70%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 75%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 80%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is greater than about 85%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 20%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 46%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 60%. In embodiments, the methacrylation efficiency of the UPVLVG alginate is 90%.

[0096] In embodiments, the methacrylation efficiency of the SLG20 alginate is less than about 70%. In embodiments, the methacrylation efficiency of the SLG20 alginate is less than about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15% or about 10%. In embodiments, the methacrylation efficiency of the SLG20 alginate is less than about 70%. In embodiments, the methacrylation efficiency of the SLG20 alginate is less than about 65%. In embodiments, the methacrylation efficiency of the SLG20 alginate is less than about 60%. In embodiments, the methacrylation efficiency of the SLG20 alginate is less than about 55%. In embodiments, the methacrylation efficiency of the SLG20 alginate is less than about 50%. In embodiments, the methacrylation efficiency of the SLG20 alginate is less than about 45%. In embodiments, the methacrylation efficiency of the SLG20 alginate is less than about 40%. In embodiments, the methacrylation efficiency of the SLG20 alginate is less than about 35%. In embodiments, the methacrylation efficiency of the SLG20 alginate is less than about 30%. In embodiments, the methacrylation efficiency of the SLG20 alginate is less than about 25%. In embodiments, the methacrylation efficiency of the SLG20 alginate is less than about 20%. In embodiments, the methacrylation efficiency of the SLG20 alginate is less than about 15%. In embodiments, the methacrylation efficiency of the SLG20 alginate is less than about 10%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 5%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 10%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 20%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 46%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 60%.

[0097] In embodiments, the methacrylation efficiency of the SLG20 alginate is between about 1% and about 80%. In embodiments, the methacrylation efficiency of the SLG20 alginate is between about 2% and about 75%, about 3% and about 70%, about 4% and about 65% or about 5% and about 60%. In embodiments, the methacrylation efficiency of the SLG20 alginate is between about 1% and about 80%. In embodiments, the methacrylation efficiency of the SLG20 alginate is between about 2% and about 75%. In embodiments, the methacrylation efficiency of the SLG20 alginate is between about 3% and about 70%. In embodiments, the methacrylation efficiency of the SLG20 alginate is between about 4% and about 65%. In embodiments, the methacrylation efficiency of the SLG20 alginate is between about 5% and about 60%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 5%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 10%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 20%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 46%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 60%.

[0098] In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 1%. In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, or about 55%. In embodiments, the methacrylation efficiency of the SLG20 alginate is greater than about 1%. In embodiments, the methacrylation efficiency of the

SLG20 alginate is greater than about 5%. In embodiments, the methacrylation efficiency of the

SLG20 alginate is greater than about 10%. In embodiments, the methacrylation efficiency of the

SLG20 alginate is greater than about 15%. In embodiments, the methacrylation efficiency of the

SLG20 alginate is greater than about 20%. In embodiments, the methacrylation efficiency of the

SLG20 alginate is greater than about 25%. In embodiments, the methacrylation efficiency of the

SLG20 alginate is greater than about 30%. In embodiments, the methacrylation efficiency of the

SLG20 alginate is greater than about 35%. In embodiments, the methacrylation efficiency of the

SLG20 alginate is greater than about 40%. In embodiments, the methacrylation efficiency of the

SLG20 alginate is greater than about 45%. In embodiments, the methacrylation efficiency of the

SLG20 alginate is greater than about 50%. In embodiments, the methacrylation efficiency of the

SLG20 alginate is greater than about 55%. In embodiments, the methacrylation efficiency of the

SLG20 alginate is 5%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 10%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 20%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 46%. In embodiments, the methacrylation efficiency of the SLG20 alginate is 60%. [0099] In embodiments, the methacrylation efficiency of the SLG100 alginate is less than about 40%. In embodiments, the methacrylation efficiency of the SLG100 alginate is less than about 35%, about 30%, about 25%, about 20%, about 15%, or about 10%. In embodiments, the methacrylation efficiency of the SLG100 alginate is less than about 40%. In embodiments, the methacrylation efficiency of the SLG100 alginate is less than about 35%. In embodiments, the methacrylation efficiency of the SLG100 alginate is less than about 30%. In embodiments, the methacrylation efficiency of the SLG100 alginate is less than about 25%. In embodiments, the methacrylation efficiency of the SLG100 alginate is less than about 20%. In embodiments, the methacrylation efficiency of the SLG100 alginate is less than about 15%. In embodiments, the methacrylation efficiency of the SLG100 alginate is less than about 10%. In embodiments, the methacrylation efficiency of the SLG100 alginate is 5%. In embodiments, the methacrylation efficiency of the SLG100 alginate is 10%. In embodiments, the methacrylation efficiency of the SLG100 alginate is 20%.

[00100] In embodiments, the methacrylation efficiency of the SLG100 alginate is between about 1% and about 40%. In embodiments, the methacrylation efficiency of the SLG100 alginate is between about 2% and about 35%, about 3% and about 30%, about 4% and about 25% or about 5% and about 20%. In embodiments, the methacrylation efficiency of the SLG100 alginate is between about 1% and about 40%. In embodiments, the methacrylation efficiency of the SLG100 alginate is between about 2% and about 35%. In embodiments, the methacrylation efficiency of the SLG100 alginate is between about 3% and about 30%. In embodiments, the methacrylation efficiency of the SLG100 alginate is between about 4% and about 25%. In embodiments, the methacrylation efficiency of the SLG100 alginate is between about 5% and about 20%. In embodiments, the methacrylation efficiency of the SLG100 alginate is 5%. In embodiments, the methacrylation efficiency of the SLG100 alginate is 10%. In embodiments, the methacrylation efficiency of the SLG100 alginate is 20%.

[00101] In embodiments, the methacrylation efficiency of the SLG100 alginate is greater than about 1%. In embodiments, the methacrylation efficiency of the SLG100 alginate is greater than about 1%, about 2%, about 3%, about 4%, about 5%, about 10% or about 15%. In embodiments, the methacrylation efficiency of the SLG100 alginate is greater than about 1%. In embodiments, the methacrylation efficiency of the SLG100 alginate is greater than about 2%. In embodiments, the methacrylation efficiency of the SLG100 alginate is greater than about 3%. In embodiments, the methacrylation efficiency of the SLG100 alginate is greater than about 4%. In embodiments, the methacrylation efficiency of the SLG100 alginate is greater than about 5%. In embodiments, the methacrylation efficiency of the SLG100 alginate is greater than about 10%. In embodiments, the methacrylation efficiency of the SLG100 alginate is greater than about 15%. In embodiments, the methacrylation efficiency of the SLG100 alginate is 5%. In embodiments, the methacrylation efficiency of the SLG100 alginate is 10%.

[00102] In embodiments, the average molecular weight of the alginate is less than about 95,000 Da. In embodiments, the average molecular weight of the alginate is less than about 65,000 Da, about 55,000 Da, about 45,000 Da, about 35,000 Da, about 25,000 Da, about 14,000 Da, about

12,000 Da, about 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da or about 2,000 Da. In embodiments, the average molecular weight of the alginate is less than about 65,000 Da. In embodiments, the average molecular weight of the alginate is less than about 55,000 Da. In embodiments, the average molecular weight of the alginate is less than about 45,000 Da. In embodiments, the average molecular weight of the alginate is less than about 35,000 Da. In embodiments, the average molecular weight of the alginate is less than about 25,000 Da. In embodiments, the average molecular weight of the alginate is less than about 14,000 Da. In embodiments, the average molecular weight of the alginate is less than about 12,000 Da. In embodiments, the average molecular weight of the alginate is less than about 10,000 Da. In embodiments, the average molecular weight of the alginate is less than about 9,000 Da. In embodiments, the average molecular weight of the alginate is less than about 8,000 Da. In embodiments, the average molecular weight of the alginate is less than about 7,000 Da. In embodiments, the average molecular weight of the alginate is less than about 6,000 Da. In embodiments, the average molecular weight of the alginate is less than about 5,000 Da. In embodiments, the average molecular weight of the alginate is less than about 4,000 Da. In embodiments, the average molecular weight of the alginate is less than about 3,000 Da. In embodiments, the average molecular weight of the alginate is less than about 2,000 Da.

[00103] In embodiments, the average molecular weight of the alginate is between about 500

Da and 50,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 40,000 Da, about 500 Da and 30,000 Da, about 500 Da and 20,000 Da, about 500 Da and 18,000 Da, about 500 Da and 16,000 Da, about 500 Da and 14,000 Da, about 500 Da and 12,000 Da, about 500 Da and 10,000 Da, about 500 Da and 9,000 Da, about 500 Da and 8,000 Da, about 500 Da and 7,000 Da, about 500 Da and 6,000 Da, about 500 Da and 5,000 Da, about 500 Da and 4,000 Da, about 500 Da and 3,000 Da, about 500 Da and 2,000 Da, or about 500 Da and 1,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 40,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 30,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 20,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 18,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 16,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 14,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 12,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 10,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 9,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 8,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 7,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 6,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 5,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 4,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 3,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 2,000 Da. In embodiments, the average molecular weight of the alginate is between about 500 Da and 1,000 Da.

[00104] In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 50,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 40,000 Da, about 1,000 Da and 30,000 Da, about 1,000 Da and 20,000 Da, about 1,000 Da and 18,000 Da, about 1,000 Da and 16,000 Da, about 1,000 Da and 14,000 Da, about 1,000 Da and 12,000 Da, about 1,000 Da and 10,000 Da, about 1,000 Da and 9,000 Da, about 1,000 Da and 8,000 Da, about 1,000 Da and 7,000 Da, about 1,000 Da and 6,000 Da, about 1,000 Da and 5,000 Da, about 1,000 Da and 4,000 Da, about 1,000 Da and 3,000 Da, or about 1,000 Da and 2,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 40,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 30,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 20,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 18,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 16,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 14,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 12,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 10,000 Da. In embodiments, the average molecular weight of the alginate is between about 1000 Da and 9,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 8,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 7,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 6,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 5,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 4,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 3,000 Da. In embodiments, the average molecular weight of the alginate is between about 1,000 Da and 2,000 Da.

[00105] In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 50,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 40,000 Da, about 2,000 Da and 30,000 Da, about 2,000 Da and 20,000 Da, about 2,000 Da and 18,000 Da, about 2,000 Da and 16,000 Da, about 2,000 Da and 14,000 Da, about 2,000 Da and 12,000 Da, about 2,000 Da and 10,000 Da, about 2,000 Da and 9,000 Da, about 2,000 Da and 8,000 Da, about 2,000 Da and 7,000 Da, about 2,000 Da and 6,000 Da, about 2,000 Da and 5,000 Da, about 2,000 Da and 4,000 Da, or about 2,000 Da and 3,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 40,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 30,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 20,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 18,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 16,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 14,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 12,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 10,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 9,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 8,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 7,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 6,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 5,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 4,000 Da. In embodiments, the average molecular weight of the alginate is between about 2,000 Da and 3,000 Da.

[00106] In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 50,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 40,000 Da, about 5,000 Da and 30,000 Da, about 5,000 Da and 20,000 Da, about 5,000 Da and 18,000 Da, about 5,000 Da and 16,000 Da, about 5,000 Da and 14,000 Da, about 5,000 Da and 12,000 Da, about 5,000 Da and 10,000 Da, about 5,000 Da and 9,000 Da, about 5,000 Da and 8,000 Da, about 5,000 Da and 7,000 Da, or about 5,000 Da and 6,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 40,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 30,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 20,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 18,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 16,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 14,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 12,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 10,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 9,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 8,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 7,000 Da. In embodiments, the average molecular weight of the alginate is between about 5,000 Da and 6,000 Da.

[00107] In embodiments, the average molecular weight of the alginate is between about 10,000 Da and 50,000 Da. In embodiments, the average molecular weight of the alginate is between about 10,000 Da and 40,000 Da, about 10,000 Da and 30,000 Da, about 10,000 Da and 20,000 Da, about 10,000 Da and 18,000 Da, about 10,000 Da and 16,000 Da, about 10,000 Da and 14,000 Da, or about 10,000 Da and 12,000 Da. In embodiments, the average molecular weight of the alginate is between about 10,000 Da and 40,000 Da. In embodiments, the average molecular weight of the alginate is between about 10,000 Da and 30,000 Da. In embodiments, the average molecular weight of the alginate is between about 10,000 Da and 20,000 Da. In embodiments, the average molecular weight of the alginate is between about 10,000 Da and 18,000 Da. In embodiments, the average molecular weight of the alginate is between about 10,000 Da and 16,000 Da. In embodiments, the average molecular weight of the alginate is between about 10,000 Da and 14,000 Da. In embodiments, the average molecular weight of the alginate is between about 10,000 Da and 12,000 Da.

[00108] In embodiments, the average molecular weight of the alginate is greater than about

500 Da. In embodiments, the average molecular weight of the alginate is greater than about 1,000 Da, about 2,000 Da, about 3,000 Da, about 4,000 Da, about 5,000 Da, about 6,000 Da, about 7,000

Da, about 8,000 Da, about 9,000 Da, about 10,000 Da, about 12,000 Da, about 14,000 Da, about 16,000 Da, about 18,000 Da, about 20,000 Da, about 30,000 Da, about 40,000 Da or about 50,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 1,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 2,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 3,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 4,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 5,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 6,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 7,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 8,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 9,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 10,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 12,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 14,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 16,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 18,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 20,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 30,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 40,000 Da.

[00109] In embodiments, the average molecular weight of the alginate is less than about 260,000 Da. In embodiments, the average molecular weight of the alginate is less than about

250,000 Da, about 240,000 Da, about 230,000 Da, about 220,000 Da, about 210,000 Da, about

200,000 Da, about 190,000 Da, about 180,000 Da, about 170,000 Da, about 160,000 Da, about

150,000 Da, about 140,000 Da, about 130,000 Da, about 120,000 Da, about 110,000 Da, about

100,000 Da, about 90,000 Da, about 80,000 Da, about 70,000 Da, about 60,000 Da, about 50,000 Da, about 40,000 Da, or about 30,000 Da. In embodiments, the average molecular weight of the alginate is less than about 260,000 Da. In embodiments, the average molecular weight of the alginate is less than about 250,000 Da. In embodiments, the average molecular weight of the alginate is less than about 240,000 Da. In embodiments, the average molecular weight of the alginate is less than about 260,000 Da. In embodiments, the average molecular weight of the alginate is less than about 250,000 Da. In embodiments, the average molecular weight of the alginate is less than about 240,000 Da. In embodiments, the average molecular weight of the alginate is less than about 230,000 Da. In embodiments, the average molecular weight of the alginate is less than about 220,000 Da. In embodiments, the average molecular weight of the alginate is less than about 210,000 Da. In embodiments, the average molecular weight of the alginate is less than about 200,000 Da. In embodiments, the average molecular weight of the alginate is less than about 190,000 Da. In embodiments, the average molecular weight of the alginate is less than about 180,000 Da. In embodiments, the average molecular weight of the alginate is less than about 170,000 Da. In embodiments, the average molecular weight of the alginate is less than about 160,000 Da. In embodiments, the average molecular weight of the alginate is less than about 150,000 Da. In embodiments, the average molecular weight of the alginate is less than about 140,000 Da. In embodiments, the average molecular weight of the alginate is less than about 130,000 Da. In embodiments, the average molecular weight of the alginate is less than about 120,000 Da. In embodiments, the average molecular weight of the alginate is less than about 110,000 Da. In embodiments, the average molecular weight of the alginate is less than about 100,000 Da. In embodiments, the average molecular weight of the alginate is less than about 90,000 Da. In embodiments, the average molecular weight of the alginate is less than about 80,000 Da. In embodiments, the average molecular weight of the alginate is less than about 70,000 Da. In embodiments, the average molecular weight of the alginate is less than about 60,000 Da. In embodiments, the average molecular weight of the alginate is less than about 50,000 Da.

[00110] In embodiments, the average molecular weight of the alginate is between about 50,000 Da and about 260,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 260,000 Da, about 75,000 Da and 250,000 Da, about 75,000 Da and 240,000 Da, about 75,000 Da and 230,000 Da, about 75,000 Da and 220,000 Da, about 75,000 Da and 210,000 Da, about 75,000 Da and 200,000 Da, about 75,000 Da and 190,000 Da, about 75,000 Da and 180,000 Da, about 75,000 Da and 170,000 Da, about 75,000 Da and 160,000 Da, about 75,000 Da and 150,000 Da, about 75,000 Da and 140,000 Da, about 75,000 Da and 130,000 Da, about 75,000 Da and 120,000 Da, about 75,000 Da and 110,000 Da, or about 75,000 Da and 100,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 260,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 250,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 240,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 230,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 220,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 210,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 200,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 190,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 180,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 170,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 160,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 150,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 140,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 130,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 120,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 110,000 Da. In embodiments, the average molecular weight of the alginate is between about 75,000 Da and 100,000 Da. [00111] In embodiments, the average molecular weight of the alginate is greater than about 50,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 60,000 Da, about 70,000 Da, about 80,000 Da, about 90,000 Da, about 100,000 Da, about 110,000

Da, about 120,000 Da, about 130,000 Da, about 140,000 Da, about 150,000 Da, about 160,000

Da, about 170,000 Da, about 180,000 Da, about 190,000 Da, about 200,000 Da, about 210,000

Da, about 220,000 Da or about 230,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 50,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 60,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 70,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 80,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 90,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 100,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 110,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 120,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 130,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 140,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 150,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 160,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 170,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 180,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 190,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 200,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 210,000 Da.

[00112] In embodiments, the average molecular weight of the alginate is less than about 350,000 Da. In embodiments, the average molecular weight of the alginate is less than about 300,000 Da, about 290,000 Da, about 280,000 Da, about 270,000 Da, about 260,000 Da, about

250,000 Da, about 240,000 Da, about 230,000 Da, about 220,000 Da, about 210,000 Da, about 200,000 Da, about 190,000 Da, or about 180,000 Da. In embodiments, the average molecular weight of the alginate is less than about 350,000 Da. In embodiments, the average molecular weight of the alginate is less than about 300,000 Da. In embodiments, the average molecular weight of the alginate is less than about 290,000 Da. In embodiments, the average molecular weight of the alginate is less than about 280,000 Da. In embodiments, the average molecular weight of the alginate is less than about 270,000 Da. In embodiments, the average molecular weight of the alginate is less than about 260,000 Da. In embodiments, the average molecular weight of the alginate is less than about 250,000 Da. In embodiments, the average molecular weight of the alginate is less than about 240,000 Da. In embodiments, the average molecular weight of the alginate is less than about 230,000 Da. In embodiments, the average molecular weight of the alginate is less than about 220,000 Da. In embodiments, the average molecular weight of the alginate is less than about 210,000 Da. In embodiments, the average molecular weight of the alginate is less than about 200,000 Da. In embodiments, the average molecular weight of the alginate is less than about 190,000 Da. In embodiments, the average molecular weight of the alginate is less than about 180,000 Da.

[00113] In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 350,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 350,000 Da, about 180,000 Da and 300,000 Da, about 180,000 Da and 290,000 Da, about 180,000 Da and 280,000 Da, about 180,000 Da and 270,000 Da, about 180,000 Da and 260,000 Da, about 180,000 Da and 250,000 Da, about 180,000 Da and 240,000 Da, about 180,000 Da and 230,000 Da, about 180,000 Da and 220,000 Da, about 180,000 Da and 210,000 Da, about 180,000 Da and 200,000 Da, or about 180,000 Da and 190,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 350,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 300,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 290,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 280,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 270,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 260,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 250,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 240,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 230,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 220,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 210,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 200,000 Da. In embodiments, the average molecular weight of the alginate is between about 180,000 Da and 190,000 Da.

[00114] In embodiments, the average molecular weight of the alginate is greater than about 180,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 190,000 Da, about 200,000 Da, about 210,000 Da, about 220,000 Da, about 230,000 Da, about 240,000 Da, about 250,000 Da, about 260,000 Da, about 270,000 Da, about 280,000 Da, about 290,000 Da, about 300,000 Da, about 310,000 Da, about 320,000 Da, or about 325,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 180,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 190,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 200,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 210,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 220,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 230,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 240,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 250,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 260,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 270,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 280,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 290,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 300,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 310,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 320,000 Da. In embodiments, the average molecular weight of the alginate is greater than about 325,000 Da.

[00115] Exemplary non-limiting combinations of alginates that may be used in the formulation of hydrogels used in the presently disclosed methods are provided in the Table 1 below. The nomenclature used in Table 1 is provided trailing in Table 2.

Table 1. Alginate Methacrylate (A1MA) Formulations

Table 2. Proposed Nomenclature for A1MA Formulations (VLV = very low viscosity; LV = low viscosity; M = methacrylation)

[00116] In some embodiments, the present methods may involve the use of hydrogels that may comprise, for example, a compound, composition and/or device provided by WO 2022/266086 A2, which is incorporated herein by reference. In some embodiments, the pharmaceutical compositions and/or devices described herein may be formulated in conjunction with a compound, composition and/or device provided by WO 2022/266086 A2. In some embodiments, the pharmaceutical compositions and/or devices described herein may be formulated in conjunction with a triazole-containing polymer, such as a triazole-containing alginate. In some embodiments, the triazole-containing alginate may comprise a compound according to any one of the following formulae:

, wherein: m and n result in a number of repeating units with a molecular weight from about 50,000 Daltons to about 500,000 Daltons.

[00117] In some embodiments, the present disclosure involves the use of a hydrogel that is a pH-responsive material, wherein the hydrogel forms upon a change in pH. In embodiments, the hydrogel forms, or is capable of flowing, when the pH is acidic, e.g., less than pH 7. In embodiments, the hydrogel forms, or is capable of forming, when the pH is basic, e.g., greater than pH 7. In embodiments, the hydrogel forms, or is capable of forming, when the pH is neutral, e.g., about pH 7. In embodiments, the hydrogel forms, or is capable of forming, when the pH is above or below the acid dissociation (Ka) or the base dissociation constant (Kb) of a moiety of the pH- responsive material.

[00118] In an aspect, the flowable material is a photo-responsive material, wherein the hydrogel forms, ceases forming, or is capable of forming, upon the introduction, removal or change in the intensity of light of a particular wavelength or a range of wavelengths, e.g., visible, UV-A, UV-B, Infrared, X-Ray, inter alia. In embodiments, the hydrogel forms, ceases forming, or is capable of forming, when the introduction of light of a particular wavelength or a range of wavelengths of a sufficient intensity triggers photopolymerization, optionally with a photo initiator.

[00119] In some embodiments, the present disclosure involves the use of a hydrogel that is a moisture-responsive material, wherein the hydrogel forms, ceases forming, or is capable of forming, upon a change in the moisture content of the material or adjacent environment. In embodiments, the polymeric components undergo self-assembly to form the resulting hydrogel in the presence of water.

[00120] Materials for assembling hydrogels may also include any provided in

Hoffman, A.S. Adv Drug Deliv Rev 2012, 64, 18-23, such as agarose, alginate (e.g., the calcium or barium salt of alginic acid), alginate-g-(alginate), carboxymethyl chitin, carrageenan, chitosan, chondroitin sulfate, collagen-acrylate, dextran, dextran sulfate, fibrin, gelatin, hyaluronic acid (HA), hyaluronic acid/glycidyl methacrylate, HA-g-NIPAAM, PAAM, P(AN-co-allyl sulfonate), P(biscarboxyl-phenoxy-phosphazene), pectin, PEG optionally with cyclodextrins (CDs), PEG-g- P(AAM-co-Vamine), PEG-bis(PLA-acrylate), PEG-PCL-PEG, , PEG-PL A-PEG, PEG-PLGA- PEG, P(PF-co-EG) optionally with acrylate groups, P(GEMA-sulfate), P(HEMA/Matrigel®), PHB, PLA-PEG-PLA, Pluronics, Pluronics and bioactive glass, P(MMA-co-HEMA), P(NIPAAM-co-AAC), P(NIPAAM-co-EMA,) PNVP, poly(D-lysine), poly(L-lysine), poly(L- lysine)/polyacrylic acid pair, P(PEG/PBO terephthalate), P(PEG-co-peptides), PPO-PEO), P(PLGA-co-serine), pullulan, and PVAC/PVA.

[00121] In some embodiments, the present disclosure involves the use of a hydrogel that is a chemically responsive material, wherein the hydrogel forms, ceases forming, or is capable of forming, upon the introduction or removal of a chemical stimulus. In embodiments, the hydrogel ceases forming upon the completion or initiation of a chemical stimulus, e.g., a chemical stimulus provided in Zhang Y.S. et al. Science. 2017, 356 (6337), eaaf3627. [00122] In some embodiments, the present disclosure involves the use of a hydrogel that can include a plurality of natural polymer macromers cross- linked with a plurality of crosslinks that are degradable after administration to a subject in vivo. The number or percentage of crosslinks linking the macromers can be varied to control the mechanical properties, swelling ratios, and degradation profiles of the hydrogels. Degradation of the crosslinks in vivo allows the hydrogel to more readily biodegrade and be used for in vivo applications. Additionally, as discussed below the photocrosslinked hydrogel can be used as a substrate for the incorporation and/or attachment of various agents and/or cells. The photocrosslinked hydrogel can be injectable and/or implantable, and can be in the form of a membrane, sponge, gel, solid scaffold, spun fiber, woven or unwoven mesh, nanoparticle, microparticle, or any other desirable configuration.

[00123] In some embodiments, the present disclosure involves the use of photocrosslinked hydrogel that includes at least on cross-link that can be hydrolyzed to allow degradation of the hydrogel in vivo. In one embodiment, the cross-link can include ester, amide, acetal, and/or ketal groups or linkages that can be readily hydrolyzed in vivo to promote degradation of the hydrogel. In one example, the hydrolyzable cross-link can include at least one hydrolyzable acrylate (e.g., methacrylate) cross-link. The hydrolyzable acrylate cross-link can include at least one hydrolyzable ester and/or hydrolyzable amide linkage. As explained further below, hydrolytic degradation of the hydrolyzable acrylate crosslink can create space for cell growth and deposition of a new extracellular matrix to replace the photocrosslinked hydrogel in vivo.

[00124] In some embodiments, the present disclosure involves the use of a photocrosslinked hydrogel that can be formed into alginate microbeads or microspheres capable of carrying and differentially and/or controllably releasing at least one bioactive agent.

[00125] In some embodiments, the present disclosure involves the use of a photocrosslinked hydrogel that is modified or configured to differentially and/or controllably release at least one bioactive agent by forming at least one concentration gradient within the hydrogel. The hydrogel can have multiple gradients in the same hydrogel, and the gradients can run in the same or opposite directions. The gradients can be comprised of different components, such as different photoalginates having different molecular weights or acrylation (e.g., methacrylation) percentages, acrylated cell adhesion ligands, bioactive factors, cells, etc. As discussed elsewhere, for example, the photocrosslinked biodegradable hydrogel can be formed into a particular shape or form to facilitate release of one or more bioactive agents according to a gradient release profile. In some aspects, one or more materials or agents can be added to the photocrosslinked biodegradable hydrogel to facilitate differential and/or controlled release of one or more bioactive agents according to a gradient release profile.

[00126] In some embodiments, the methods of the present disclosure involve the use of a tissue mimicking composition comprising a hydrogel with at least one channel or lumen. In some embodiments, the methods of the present disclosure involve the use of a hydrogel with at least one open channel. In some embodiments, the methods of the present disclosure involve the use of a hydrogel with a first tubular channel or lumen and a second tubular channel or lumen. In certain aspects, the first and second tubular channel or lumen each can include a horizontal segment that intersects more than one layer of the bulk hydrogel matrix. The second tubular channel or lumen can interpenetrate the first channel or lumen where interpenetrating is defined as the spatial relationship between two channels or lumens wherein one channel or lumen intersects at least once, a plane between two separate portions of the other channel. The tubular channels or lumens can also be branched. For example, the tubular channels or lumens may branch, as observed in the torus knot model, wherein the tubular channels or lumens reconverge at another point within the hydrogel. However, branched structures can also include channels or lumens which extend from the first tubular channel or lumen and/or the second tubular channel or lumen and terminate within the hydrogel. For example, tree-like structures can be designed and produced using the present approach. In certain embodiments, the tubular channels or lumens have a diameter of 300 to 500 microns, 500 microns or less, 400 microns or less, or 300 microns or less. The tubular channel or lumens can also be perfusable. In addition, the tubular channels or lumens can also be expandable in response to increases in pressure therein. Tubular channels or lumens can be lined with cells, including epithelial and endothelial cells. In certain aspects, the first tubular channel or lumen is lined with endothelial cells. In certain aspects, the second tubular channel or lumen is lined with epithelial cells.

[00127] The first tubular channel can also include a first tubular inlet and a first tubular outlet on the surface of the hydrogel matrix. The second tubular channel can also include a second tubular inlet and a second tubular outlet on the surface of the hydrogel matrix. The first tubular channel can include a valve or other positive feature. Tubular channels can also include spikes that extend therefrom into the hydrogel matrix. Tubular channels can be filled with any appropriate fluid or gas. Such fluids or gases can include, by way of example but not limitation, bodily fluids and oxygen. In certain aspects, the first tubular channel can be filled with a fluid.

[00128] In certain other aspects, the first tubular channel can be filled with culture media, red blood cells, blood, lymphatic cells, urine, bile and/or gases such as nitrogen and/or oxygen. In certain aspects, the second tubular channel can be filled with culture media, red blood cells, blood, lymphatic cells, urine, bile and/or gases such as nitrogen and/or oxygen. Tubular channels can also be filled with one or more different fluids and/or gases.

[00129] Tissue mimicking compositions used in the present methods may comprise hydrogels that include more than two tubular channels. For example, a hydrogel can include a third tubular channel and a fourth tubular channel. A tubular channel can interpenetrate more than one other tubular channel. For instance, a third tubular channel can interpenetrate a fourth tubular channel. Similarly, a second tubular channel can interpenetrate a first tubular channel and a third tubular channel. Tubular channel networks comprising multiple tubular channels may also interpenetrate at least one tubular channel or at least one other tubular channel network. For example, a third tubular channel may interpenetrate a first tubular channel that is also interpenetrated by a second tubular channel. As another example, a third tubular channel and fourth tubular channel can be interpenetrating and interpenetrate a first tubular channel or an interpenetrating network comprising a first tubular channel and a second tubular channel. In this manner, complex models can be constructed which permit complex interactions between tubular channels and tubular channel networks. The foregoing examples of multiple tubular channels are for exemplary purposes only and not intended to limit this disclosure.

[00130] It should be noted that list of polymers for various features of the present devices may overlap, as potential modifications to any given polymer can render it useful for to form the channel or a semi-permeable biomaterial. A semi-permeable biomaterial portion serves many purposes, including containing pro-angiogenic compounds, oxygen-releasing compounds, immune-modulating compounds, or other biologically active compounds, as well as cells, including endothelial cells. A semi-permeable biomaterial can affect the local site where it is implanted, including but not limited to vascularization, immunomodulation, and controlled release of other compounds, and/or confer useful advantages to the channel portion of the device through these and other related means.

[00131] 3D Printable Compositions [00132] The inventors have previously described a 3D printing process that can fabricate 3D engineered tissues with biologically-inspired design criteria including, but not limited to, conforming to Murray's Law, multiscale branched vessels from tens to hundreds of micrometers in diameter, smooth inner walls, circular cross sections, and multiple inlet/outlets. Indeed, with printing parameter optimization, the limit to what can be fabricated depends on what one can model. In addition, more complex designs that contain heterogenous properties in a single layer or in multiple layers can be fabricated. For instance, grayscale photomasks with predefined gradients can be incorporated to obtain a layer with varied stiffness or for controlled immobilization of biomolecules and cells while still using the same vat and solution. Additionally, utilization of fractal space-filling models to computationally grow tissue networks around and through preexisting tissue networks or following the architecture of native tissues can be achieved by computer growth models for even more complex and physiologically relevant 3D models. These mathematical fractal, space-filling models can be derived from, for example, knot theory, the Hilbert curve, and the L-system. Such mathematical fractal space-filling models to predict idealized tissue or vessel networks include, but are not limited to knot theory, Plumber's Nightmare, Peano curve, Hilbert curve, Pythagoras tree, and Brownian tree models. As an example, the Plumber's Nightmare model essentially comprises two Vascular Ladder models that are connected to each other by straight vertical cylinders. Multiple Plumber's Nightmare models can be intercalated such that they are interpenetrating. The Vascular Ladder models are comprised of 1 inlet and 1 outlet with two horizontal cylinders that are connected by diagonal cylinders, resulting in interchannel junctions.

[00133] Photopolymerizable hydrogel materials such as poly(ethylene glycol) diacrylate (PEGDA) can be crosslinked using a photoinitiator system such as lithium acylphosphinate (LAP) (Fairbanks et al. 2009) which absorbs in the UV to visible light wavelength range. By adding, for example, low concentrations of carbon black (which can absorb light across all UV-visible light spectrum), or low concentrations of tartrazine (which has a peak light absorption near 500 nm), the inventors can limit the depth of penetration of light. To quantify this process, the inventors developed a photorheology assay to monitor hydrogels polymerization and stiffness evolution as a function of light dosage and sample thickness. Indeed, the addition of additive materials, such as tartrazine, control the extent of gelation of the prepolymerization mixture by impacting the gelation kinetics and final gel rheological properties. Other materials include -ene modified natural and synthetic materials that can be photopolymerized such as alginate, silk, dextran, chondroitin sulfate, hyaluronic acid, cellulose, heparin, and poly(caprolactone) and multi-component versions of these.

[00134] To achieve complex patterning of multilayered hydrogels, on the order of several centimeters, with high pattern fidelity, light exposure during the printing process is controlled so that the light projected onto the build platform interacts mainly with the layer that undergoes gelation for either partial or complete gelation. Radical mediated photopolymerization of hydrogels utilizes a photoinitiator — a molecule sensitive to a particular wavelength range that, upon light absorption, the molecule decays and release free radicals which can catalyze hydrogel polymerization. To this end, it is imperative to quantify the wavelength sensitivity of the photoinitiator. High concentrations of photoinitiator will absorb more light and provide higher z- resolution by limiting penetration depth of the incident light. However, high photoinitiator concentrations disrupt the photopolymerization reaction (more free radicals have a higher chance of annihilating each other), and photoinitiators at high concentrations are cytotoxic. In addition, with high x-y resolution from the projector, a complication is that light shines through the z- direction of the previously printed layers, potentially limiting the ability to form complex overhang structures (such as found in tissues, such as vasculature), and also may cause phototoxicity to entrapped cells.

[00135] Thus, to achieve high resolution printing, the inventors have identified photochemical means to provide high z-resolution in bioprinted tissues while maintaining high cell viability. To address the concerns outlined above, the inventors have identified a general strategy whereby biocompatible materials or chemicals are added to the prepolymerization solution to provide higher z-resolution. The additive material is selected based on three criteria: 1) ability to absorb light wavelengths which fully encompass the photosensitive wavelength range of the photoinitiator, 2) limited participation or limited inhibition of photopolymerization reactions, and 3) biocompatibility at the concentrations desired. This additive material is referred to herein as a biocompatible, light-absorbing additive material suitable to control light penetration. Multiple molecules have been screened that absorb light, limiting the penetration depth of light into already formed layers. Suitable molecules absorb in the same region as the photoinitiator used in the prepolymerization solution. Examples of molecules capable of controlling light penetration and therefore suitable for use as the biocompatible, light-absorbing additive material include carbon black, yellow food coloring, tartrazine, nanoparticles, microparticles, gold nanoparticles, riboflavin, phenol red, Beta-carotene, curcumin, saffron, and turmeric. Proteins may also act as suitable biocompatible, light-absorbing additive materials provided that their peak absorption overlaps with the peak absorption of the photoinitiator and matched to the incident light source. Additionally, the inventors recognize that cells that are transfected or transduced with proteins that absorb in the same region as the photoinitiator, such as cyan fluorescent protein (CFP) or green fluorescent protein (GFP), can be used at high concentrations, with reduced or no additives, to result in reduced lateral overcuring due to the light absorbing molecules present inside cells. Additionally, the inventors’ methodology allows us to print hydrogels with both horizontal and vertical channels due to stringent control of the penetration of the projected light.

[00136] To address potential cell viability concerns associated with long print times for fabrication of engineered tissues with this method, the cell viability can be maintained or enhanced in multiple ways. One method involves lowering the metabolic activity of the cells by printing in hypothermic conditions. Similar to hypothermic preservation of solid organs for extended preservation of transplant organs (typically done at 4 °C), decreasing the temperature in which cells are printed in will significantly decrease cellular metabolism. For example, the inventors have demonstrated that a cold room (4 °C) can be utilized to achieve hypothermic bioprinting. Additionally, incorporation of vitamins, growth factors, or serum in the prepolymerization solution, for readily accessible supply of nutrients, can be done to maintain or enhance cell viability. Importantly, photopolymerization is highly tolerant of decreased temperatures with only a modest increase in required exposure time. Further, decreasing the temperature of the entire 3D printing apparatus and reagents may help to quench heat generated from incident light or the photopolymerization process. In addition, molecules such as PEG and glycerol have been widely used as cryoprotectants in cell culture. Thus, use of a polymer such as PEG during the hypothermic printing process may enhance cell survivability.

[00137] Branching multi-scale transport systems are found in all multicellular life. Similar to the highly complex branching structure of vascular networks, the respiratory tree is also composed of a complex branching structure for sufficient supply of air in the distal regions of the lung. It has been indicated that endothelial cells may aid in lung epithelial branching. However, current manufacturing techniques do not allow for structures that mimic the anatomical complexity of native lung tissue. By using 3D printing, it should be possible to produce structures that mimic the anatomical complexity of native lung tissue and vasculature. The proposed approach can allow the printing of such structures and for embedding endothelial and epithelial cell types in channel lumens to mimic vascular and respiratory networks. The circular cross-sections attainable permit the development of confluent cell layers along the channel lumens. Given the higher z-resolution under the proposed approach, the channels can more closely mimic vascular and respiratory networks. The disclosed methods and materials can enable the fine control of the geometry and architecture of multiple networks. By using fractal, space-filling models akin to physiological vascular networks, the technology permits the design and fabrication of relevant 3D constructs with interpenetrating channels.

[00138] Additionally, the proposed approach can be combined with other scaffold fabrication techniques, such as porogen leaching or surface coating, to result in physiologically relevant complex constructs with modified internal microarchitecture or surface properties. Additionally, the proposed approach can be used for fabrication of microfluidic devices for organ- on-a-chip or human-on-a-chip applications. Additionally, the printer can be modified to include specific sensors for ensuring printing of more precise layer thickness.

[00139] In certain embodiments, a prepolymerization solution is provided. The prepolymerization solution comprises a photosensitive polymer having a molecular weight greater than 2,000 Daltons, a photoinitiator, and a biocompatible, light-absorbing additive material suitable to control light penetration. In some embodiments, the prepolymerization solution can also include one or more living cells (referred to herein simply as a cell). In some aspects, the prepolymerization solution can include a cryoprotectant such as low molecular weight PEG, glycerol, ethylene glycol, sucrose, propylene glycol, trehalose, raffinose, guar gum, xanthan gum, and D-mannitol. Cryoprotectants can be permeating or non-permeating. Each of these components is described in further detail herein. The prepolymerization solution can also include a water content of 10 wt % to about 99.5 wt %. The prepolymerization solution can include a water content of 80 wt % to about 90 wt %. In some aspects, the prepolymerization can further comprise DMEM media, serum, proteins, growth factors, thickening agents and/or anti-clumping components. Such components can provide nutrition for and/or neutral buoyancy for cells in the prepolymerization solution.

[00140] In certain embodiments, a photosensitive polymer having a molecular weight greater than 2,000 Daltons can be used. Photosensitive polymers can include at least two vinyl groups per molecule of polymer. Such vinyl groups can include acrylate, acrylamide and methacrylate. Photosensitive polymers which can be used include, by example but not limitation, poly(ethylene glycol) diacrylate (PEDGA), cell-adhesive poly(ethylene glycol), MMP-sensitive poly(ethylene glycol), poly(ethylene glycol) dimethacrylate (PEGDMA), poly(ethylene glycol) diacrylamide (PEGDAAm), gelatin methacrylate (GelMA), methacrylated hyaluronic acid (MeHA), and PEGylated fibrinogen. The photosensitive polymers can also be modified by the conjugation of cell-adhesive peptides such asCGRGDS.

[00141] In some embodiments, tissue mimicking compositions for use in the presently disclosed embodiments are printed using additive manufacturing technology such as 3D printing using inks comprising any of the polymers described elsewhere in this section. In some embodiments, the inks are biocompatible. The composition of the ink (that is, the ratio of components, such as polymer components) may, without being bound by theory, contribute to the favorable properties of the tissue mimicking compositions described herein. In some embodiments, the ink comprises gelatin methacrylate (GelMA). In some compositions, the ink comprises polyethylene glycol diacrylate (PEGDA). In some embodiments, the ink comprises GelMA and PEGDA. In some embodiments, the ink used to print the presently disclosed compositions is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20% GelMA by weight, or any range derivable therein. In some embodiments, the ink used to print the presently disclosed compositions is about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% GelMA by weight, or any range derivable therein. In some embodiments, the ink used to print the presently disclosed compositions is about 10% GelMA by weight. In some embodiments, the ink used to print the presently disclosed compositions is about 0.5%, about 0.75%, about 1%, about 1.25%, about 1.5%, about 1.75%, about 2%, about 2.25%, about 2.5%, about 2.75%, about 3%, about 3.25%, about 3.5%, about 3.75%, about 4%, about 4.25%, about 4.5%, about 4.75%, about 5%, about 5.25% about 5.5%, about 5.75%, or about 6%, PEGDA by weight, or any range derivable therein. In some embodiments, the ink used to print the presently disclosed compositions is about 2%, about 2.25%, about 2.5%, about 2.75%, about 3%, about 3.25%, about 3.5%, about 3.75%, or about 4% PEGDA by weight, or any range derivable therein. In some embodiments, the ink used to print the presently disclosed compositions is about 3.25% PEGDA by weight. In some embodiments, the printed composition is exposed to light. The amount of time that the printed composition is exposed to light will vary based on the ink composition and may vary to optimize the characteristics or properties of the composition. The amount of time that the printed composition is exposed to light may be selected to optimize or facilitate production of the presently disclosed tissue mimicking compositions. In some embodiments, the printed composition is exposed to light for about 0.5 seconds, about 1 second, about 1.5 seconds, about 2 seconds, about 2.5 seconds, about 3 seconds, about 3.5 seconds, about 4 seconds, about 4.5 seconds, about 5 seconds, about 5.5 seconds, about 6 seconds, about 6.5 seconds, about 7 seconds, about 7.5 seconds, about 8 seconds, about 8.5 seconds, about 9 seconds, about 9.5 seconds, about 10 seconds, or any range derivable therein. In some embodiments, the printed composition is exposed to light for about 4 seconds, about 4.5 seconds, about 5 seconds, about 5.5 seconds, about 6 seconds, about 6.5 seconds, about 7 seconds, about 7.5 seconds, about 8 seconds, or any range derivable therein. In some embodiments, the printed composition is exposed to light for about 6 seconds. The light intensity may be selected to optimize the characteristics or properties of the composition. The light intensity may be selected to optimize or facilitate production of the presently disclosed tissue mimicking compositions. The light intensity may be about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, or about 55%. The light intensity in some embodiments is about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40%. In some embodiments, the light intensity is about 38%.

[00142] In certain embodiments, a photoinitiator can be used. The photoinitiator is a molecule sensitive to a particular wavelength range that, upon light absorption, the molecule decays and releases free radicals which can catalyze hydrogel polymerization. Such photoinitiators can include, by way of example but not limitation, lithium acylphosphinate, Irgacure 2959, the Eosin Y system (consisting of eosin Y, l-vinyl-2-pyrrolidinone (NVP), and triethanolamine (TEA)), tris(triphenlphosphine)ruthenium(II), and camphorquinone which is typically used with either ethyl 4-N,N-dimethylaminobenzoate or TEA and the photosensitizer isopropyl thioxanthone. High concentrations of photoinitiators can be used to achieve increased z-resolution by limiting the penetration depth of incident light, however, these high concentrations can disrupt the photopolymerization reaction and are cytotoxic. In addition, because light can shine through previously printed layers, use of high concentrations of photoinitiators can limit the ability to print complex overhang structures and cause phototoxicity to entrapped cells.

[00143] In certain embodiments a biocompatible, light-absorbing additive material can be used. The biocompatible, light-absorbing additive material is selected based on three criteria: 1) ability to absorb light wavelengths which fully encompass the photosensitive wavelength range of the photoinitiator, 2) limited participation or limited inhibition of photopolymerization reactions, and 3) biocompatibility at the concentrations desired. The biocompatible, light-absorbing additive material can be organic. The biocompatible, light-absorbing additive material can include, by way of example, but not limitation carbon black, yellow food coloring, tartrazine, nanoparticles, microparticles, gold nanoparticles, riboflavin, phenol red, Beta-carotene, curcumin, saffron, and turmeric. Additionally, cells that are transfected or transduced with proteins that absorb in the same region as the photoinitiator, such as cyan fluorescent protein (CFP) or green fluorescent protein (GFP), can be used at high concentrations, with reduced or no additives, to result in reduced lateral overcuring due to the light absorbing molecules present inside cells. The biocompatible, lightabsorbing additive material enables printing of hydrogels with both horizontal and vertical channels due to stringent control of the penetration of the projected light which allows for high z- resolution as the penetration of light into already printed layers is reduced.

[00144] In some embodiments, a hydrogel matrix is provided. The hydrogel matrix can include a first tubular channel and a second tubular channel. The hydrogel matrix can be porous. The hydrogel matrix can also include a first cell type and a second cell type embedded therein. In certain aspects, the hydrogel matrix can include a first cell type embedded therein.

[00145] The hydrogel matrix can be produced in one or more layers and in certain embodiments, will include more than 1,000 layers, from about 10 layers to about 2,000 layers, from about 10 layers to about 1,000 layers, from about 10 to about 500 layers, from about 10 to about 100 layers, from about 100 to about 2,000 layers, from about 100 to about 1,000 layers, from about 100 layers to 500 layers, from about 100 layers to about 300 layers, from about 500 layers to about 1,000 layers, from about 500 layers to about 2,000 layers, from about 1 ,000 layers to about 2,000 layers, and any range therebetween of the above. In certain embodiments, each layer can have a thickness of from about 25 microns to about 100 microns, from about 50 microns to about 100 microns, from about 25 microns to about 50 microns, and any range therebetween. In certain other aspects, each layer can have a thickness of 50 microns. In still other aspects, each layer can have a thickness of less than 50 microns. Tn some embodiments, each layer can have a thickness of 25 microns. In certain aspects, each layer can have a thickness of 100 microns. In certain aspects, the one or more layers of the hydrogel matrix can include a first cell type wherein one or more other layers of the hydrogel matrix include a second cell type, but not the first cell type.

[00146] The one or more layers of the hydrogel matrix can have cells embedded therein. In certain aspects, the one or more layers of the hydrogel matrix adjacent to the one or more layers of the hydrogel matrix with embedded cells comprises an extracellular matrix protein.

[00147] The one or more layers of the hydrogel matrix can be formed from a photosensitive polymer. In certain aspects, the one or more layers of the hydrogel matrix can be formed from a second photosensitive polymer. The one or more layers of the hydrogel matrix can each include a first portion and second portion. In certain aspects, the first portion is formed from the photosensitive polymer and the second portion is formed from a second photosensitive polymer having a molecular weight of greater than 2,000 Daltons. In certain aspects, the first portion can include a first cell type embedded therein and the second portion can include a second cell type embedded therein, wherein the first cell type is different from the second cell type. In certain aspects, the first portion can include a first fluorophore and the second portion can include a second fluorophore, wherein the first fluorophore is different from the second fluorophore.

[00148] In some embodiments, a hydrogel can include a first tubular channel and a second tubular channel. In certain aspects, the first and second tubular channel each can include a horizontal segment that intersects more than one layer of the bulk hydrogel matrix. The second tubular channel can interpenetrate the first channel where interpenetrating is defined as the spatial relationship between two channels wherein one channel intersects at least once, a plane between two separate portions of the other channel. The tubular channels can also be branched. For example, the tubular channels may branch, as observed in the torus knot model, wherein the tubular channels reconverge at another point within the hydrogel. However, branched structures can also include channels which extend from the first tubular channel and/or the second tubular channel and terminate within the hydrogel. For, example, tree-like structures can be designed and produced using the present approach. In certain embodiments, the tubular channels have a diameter of 300 to 500 microns, 500 microns or less, 400 microns or less, or 300 microns or less. The tubular channel can also be perfusable. In addition, the tubular channels can also be expandable in response to increases in pressure therein. Tubular channels can be lined with cells, including epithelial and endothelial cells. In certain aspects, the first tubular channel is lined with endothelial cells. In certain aspects, the second tubular channel is lined with epithelial cells.

[00149] The first tubular channel can also include a first tubular inlet and a first tubular outlet on the surface of the hydrogel matrix. The second tubular channel can also include a second tubular inlet and a second tubular outlet on the surface of the hydrogel matrix. The first tubular channel can include a valve or other positive feature. Tubular channels can also include spikes that extend therefrom into the hydrogel matrix. Tubular channels can be filled with any appropriate fluid or gas. Such fluids or gases can include, by way of example but not limitation, bodily fluids and oxygen. In certain aspects, the first tubular channel can be filled with a fluid.

[00150] In certain other aspects, the first tubular channel can be filled with culture media, red blood cells, blood, lymphatic cells, urine, bile and/or gases such as nitrogen and/or oxygen. In certain aspects, the second tubular channel can be filled with culture media, red blood cells, blood, lymphatic cells, urine, bile and/or gases such as nitrogen and/or oxygen. Tubular channels can also be filled with one or more different fluids and/or gases.

[00151] Hydrogels of the present disclosure can include more than two tubular channels. For example, a hydrogel can include a third tubular channel and a fourth tubular channel. A tubular channel can interpenetrate more than one other tubular channel. For instance, a third tubular channel can interpenetrate a fourth tubular channel. Similarly, a second tubular channel can interpenetrate a first tubular channel and a third tubular channel. Tubular channel networks comprising multiple tubular channels may also interpenetrate at least one tubular channel or at least one other tubular channel network. For example, a third tubular channel may interpenetrate a first tubular channel that is also interpenetrated by a second tubular channel. As another example, a third tubular channel and fourth tubular channel can be interpenetrating and interpenetrate a first tubular channel or an interpenetrating network comprising a first tubular channel and a second tubular channel. In this manner, complex models can be constructed which permit complex interactions between tubular channels and tubular channel networks. The foregoing examples of multiple tubular channels are for exemplary purposes only and not intended to limit this disclosure. [00152] It should be noted that list of polymers for various features of the present devices may overlap, as potential modifications to any given polymer can render it useful for to form the channel or the semi-permeable biomaterial. The semi-permeable biomaterial portion serves many purposes, including containing pro-angiogenic compounds, oxygen-releasing compounds, immune-modulating compounds, or other biologically active compounds, as well as cells, including endothelial cells. The semi-permeable biomaterial can affect the local site where it is implanted, including but not limited to vascularization, immunomodulation, and controlled release of other compounds, and/or confer useful advantages to the channel portion of the device through these and other related means.

[00153] Cells

[00154] In some embodiments, the presently disclosed compositions can include a cell or tissue, e.g., a living cell or tissue, which in some embodiments is encapsulated in, or coated with, a polymer. In such embodiments, the surface of the polymer encapsulation or coating is modified with moieties or compounds disclosed herein. In some embodiments, the cell can include an exogenous nucleic acid that encodes a therapeutic or diagnostic polypeptide.

[00155] The cell type chosen for inclusion in the disclosed compositions depends on the desired therapeutic effect. The cells may be from the patient (autologous cells), from another donor of the same species (allogeneic cells), or from another species (xenogeneic). Xenogeneic cells are easily accessible, but the potential for rejection and the danger of possible transmission of viruses to the patient restricts their clinical application. Any of these types of cells can be from natural sources, stem cells, derived cells, or genetically engineered cell.

[00156] In some embodiments, the cell is a genetically engineered cell that secretes a therapeutic agent, such as a protein or hormone for treating a disease or other condition. In some embodiments, the cell is a genetically engineered cell that secretes a diagnostic agent. In some embodiments, the cell is a stem cell, e.g., an embryonic stem cell, mesenchymal stem cell, hepatic stem cell, or bone marrow stem cell.

[00157] Types of cells for inclusion in the disclosed compositions include cells from natural sources, such as cells from xenotissue, cells from a cadaver, and primary cells; stem cells, such as embryonic stem cells, mesenchymal stem cells, and induced pluripotent stem cells; derived cells, such as cells derived from stem cells, cells from a cell line, reprogrammed cells, reprogrammed stem cells, and cells derived from reprogrammed stem cells; and genetically engineered cells, such as cells genetically engineered to express a protein or nucleic acid, cells genetically engineered to produce a metabolic product, and cells genetically engineered to metabolize toxic substances. [00158] Types of cells for inclusion in the disclosed compositions include liver cells (e.g., hepatoblasts liver stellate cells, biliary cells, or hepatocytes), insulin producing cells (e.g., pancreatic islet cells, isolated pancreatic beta cells, or insulinoma cells), kidney cells, epidermal cells, epithelial cells, neural cells, including neurons and glial cells (e.g., astrocytes), ganglion cells, retinal epithelial cells, adrenal medulla cells, lung cells, cardiac muscle cells, osteoblast cells, osteoclast cells, bone marrow cells, spleen cells, thymus cells, glandular cells, blood cells (e.g., T cells, B cells, macrophage lineage cells, lymphocytes, or monocytes), endocrine hormone- producing cells (e.g., parathyroid, thyroid, or adrenal cells), cells of intestinal origin and other cells acting primarily to synthesize and secret or to metabolize materials, endothelial cells (e.g., capillary endothelial cells), fibroblasts (e.g., dermal fibroblasts), myogenic cells, keratinocytes, smooth muscle cells, progenitor cells (e.g., bone marrow progenitor cells, adipose progenitor cells, hepatic precursor cells, endothelia progenitor cells, peripheral blood progenitor cells, or progenitor cells from muscle, skin ) marrow stromal cells cell lines (e.g., CHO cells, MDCK cells and PC12 cells).

[00159] A particular cell type is a pancreatic islet cell or other insulin-producing cell. Hormone-producing cells can produce one or more hormones, such as insulin, parathyroid hormone, anti-diuretic hormone, oxytocin, growth hormone, prolactin, thyroid stimulating hormone, adrenocorticotropic hormone, follicle-stimulating hormone, lutenizing hormone, thyroxine, calcitonin, aldosterone, Cortisol, epinephrine, glucagon, estrogen, progesterone, and testosterone. Genetically engineered cells are also suitable for inclusion in the disclosed devices. In some embodiments, the cells are engineered to produce one or more hormones, such as insulin, parathyroid hormone, antidiuretic hormone, oxytocin, growth hormone, prolactin, thyroid stimulating hormone, adrenocorticotropic hormone, follicle-stimulating hormone, lutenizing hormone, thyroxine, calcitonin, aldosterone, Cortisol, epinephrine, glucagon, estrogen, progesterone, and testosterone. In some embodiments, the cells are engineered to secrete blood clotting factors (e.g., for hemophilia treatment) or to secrete growth hormones. In some embodiments, the cells are contained in natural or bioengineered tissue. For example, the cells for inclusion in the disclosed devices are in some embodiments a bioartificial renal glomerulus. In some embodiments, the cells are suitable for transplantation into the central nervous system for treatment of neurodegenerative disease. [00160] In certain embodiments, a cell may be included in the prepolymerization solution and/or hydrogel. Such cells can include endothelial and epithelial cell types. Such cells can be human mesenchymal stem cells (hMSCs). By way of example but not limitation, epithelial cells can include human bronchial epithelial cells HBECs), columnar ciliated epithelial cells, mucous cells, serous cells, basal cells, Clara cells, neureoendocrme cells, type I and type II alveolar cells, and A549 adenocarcinomic human alveolar basal epithelial cells. By way of example but not limitation, epithelial cells can be characterized based on staining for E-cadherin, surfactant proteins A, C and D, basal marker keratin 14, and TTF-1 Similarly, by way of example but not limitation, endothelial cells can include human umbilical vein endothelial cells HUVECs), human pulmonary microvascular endothelial cells, and induced pluripotent endothelial cells. By way of example but not limitation, endothelial cells can be characterized based on statining for platelet endothelial cell adhesion molecular (PECAM/CD31) and vascular endothelial (VE)-cadherin.

[00161] Cells can be obtained directly from a donor, from cell culture of cells from a donor, or from established cell culture lines. In particular embodiments, cells are obtained directly from a donor, washed and implanted directly in combination with the polymeric material. The cells are cultured using techniques known to those skilled in the art of tissue culture.

[00162] Cell viability can be assessed using standard techniques, such as histology and fluorescent microscopy. The function of the implanted cells can be determined using a combination of these techniques and functional assays. For example, in the case of hepatocytes, in vivo liver function studies can be performed by placing a cannula into the recipient's common bile duct. Bile can then be collected in increments. Bile pigments can be analyzed by high pressure liquid chromatography looking for underivatized tetrapyrroles or by thin layer chromatography after being converted to azodipyrroles by reaction with diazotized azodipyrroles ethylanthranilate either with or without treatment with P-glucuronidase. Diconjugated and monoconjugated bilirubin can also be determined by thin layer chromatography after alkalinemethanolysis of conjugated bile pigments. In general, as the number of functioning transplanted hepatocytes increases, the levels of conjugated bilirubin will increase. Simple liver function tests can also be done on blood samples, such as albumin production. Analogous organ function studies can be conducted using techniques known to those skilled in the art, as required to determine the extent of cell function after implantation. For example, pancreatic islet cells and other insulin-producing cells can be implanted to achieve glucose regulation by appropriate secretion of insulin. Other endocrine tissues and cells can also be implanted.

[00163] The site, or sites, where cells are to be implanted is determined based on individual need, as is the requisite number of cells. For cells replacing or supplementing organ or gland function (for example, hepatocytes or islet cells), the mixture can be injected into the mesentery, subcutaneous tissue, retroperitoneum, preperitoneal space, and intramuscular space.

[00164] The amount and density of cells included in the disclosed devices will vary depending on the choice of cell and site of implantation. In some embodiments, the single cells are present in the hydrogel capsule at a concentration of 0.1 x 10⁶to 4 x 10⁶ cells/ml, more particularly 0.5 x 10⁶ to 2 x 10⁶ cells/ml. In other embodiments, the cells are present as cell aggregates. For example, islet cell aggregates (or whole islets) preferably contain about 1500-2000 cells for each aggregate of 150pm diameter, which is defined as one islet equivalent (IEQ). Therefore, in some embodiments, islet cells are present at a concentration of 100-10000 lE/ml, particularly 200-3,000 lE/ml, more particularly 500- 1500 lEQ/ml.

[00165] 1. Islet Cells and Other Insulin-Producing Cells

[00166] In particular embodiments, the disclosed compositions contain islet cells or other insulin-producing cells. Methods of isolating pancreatic islet cells are known in the art. Field et al., Transplantation 61 : 1554 (1996); Linetsky et al., Diabetes 46: 1 120 (1997). Fresh pancreatic tissue can be divided by mincing, teasing, comminution and/or collagenase digestion. The islets can then be isolated from contaminating cells and materials by washing, filtering, centrifuging or picking procedures. Methods and apparatus for isolating and purifying islet cells are described in U.S. Patent Nos. 5,447,863, 5,322,790, 5,273,904, and 4,868,121. The isolated pancreatic cells may optionally be cultured prior to inclusion in the hydrogel capsule using any suitable method of culturing islet cells as is known in the art. See e.g., U.S. Patent No. 5,821,121. Isolated cells may be cultured in a medium under conditions that helps to eliminate antigenic components. Insulinproducing cells can also be derived from stem cells and cell lines and can be cells genetically engineered to produce insulin.

[00167] 2. Genetically Engineered Cells

[00168] In some embodiments, the disclosed compositions contain cells genetically engineered to produce a protein or nucleic acid (e.g., a therapeutic protein or nucleic acid). In these embodiments, the cell can be, for example, a stem cell (e.g., pluripotent), a progenitor cell (e.g., multipotent or oligopotent), or a terminally differentiated cell (i.e., unipotent). Any of the disclosed cell types can be genetically engineered. The cell can be engineered, for example, to contain a nucleic acid encoding, for example, a polynucleotide such miRNA or RNAi or a polynucleotide encoding a protein. The nucleic acid can be, for example, integrated into the cells genomic DNA for stable expression or can be, for example, in an expression vector (e.g., plasmid DNA). The polynucleotide or protein can be selected based on the disease to be treated (or effect to be achieved) and the site of transplantation or implantation. In some embodiments, the polynucleotide or protein is anti -neoplastic. In other embodiments, the polynucleotide or protein is a hormone, growth factor, or enzyme.

[00169] In some embodiments, the engineered cell can be a cell isolated or obtained from the retina. In some embodiments, the engineered cell can be a pigment epithelial cell, such as a retinal pigment epithelial cell (e.g., ARPE-19). In some embodiments, the engineered cell can form clusters of engineered cells.

[00170] 3. Non-Cellular Materials

[00171] In some embodiments, the cells secrete a therapeutically effective substance, such as a protein or nucleic acid. In some embodiments, the cells produce a metabolic product. In some embodiments, the cells metabolize toxic substances. In some embodiments, the cells form structural tissues, such as skin, bone, cartilage, blood vessels, or muscle. In some embodiments, the cells are natural, such as islet cells that naturally secrete insulin, or hepatocytes that naturally detoxify. In some embodiments, the cells are genetically engineered to express a heterologous protein or nucleic acid and/or overexpress an endogenous protein or nucleic acid. In some embodiments, the cells are genetically engineered to produce a new or different product, which can be an expression product of the engineered gene(s) or another product, such as a metabolite, produced because of the engineered gene(s).

[00172] Therapeutic agents that can be included in the device, or engineered into cells included in the device include, for example, thyroid stimulating hormone; beneficial lipoproteins such as Apol; prostacyclin and other vasoactive substances, anti-oxidants and free radical scavengers; soluble cytokine receptors, for example soluble transforming growth factor (TGF) receptor, or cytokine receptor antagonists, for example ILlra; soluble adhesion molecules, for example ICAM-1 ; soluble receptors for viruses, e.g., CD4, CXCR4, CCR5 for HIV; cytokines; elastase inhibitors; bone morphogenetic proteins (BMP) and BMP receptors 1 and 2; endoglin; serotonin receptors; tissue inhibiting metalloproteinases; potassium channels or potassium channel modulators; anti-inflammatory factors; angiogenic factors including vascular endothelial growth factor (VEGF), transforming growth factor (TGF), hepatic growth factor, and hypoxia inducible factor (HIF); polypeptides with neurotrophic and/or anti-angiogenic activity including ciliary neurotrophic factor (CNTF), glial-derived neurotrophic factor (GDNF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3, nurturin, fibroblast growth factors (FGFs), endostatin, ATF, fragments of thrombospondin, variants thereof and the like. More particular polypeptides are FGFs, such as acidic FGF (aFGF), basic FGF (bFGF), FGF-1 and FGF- 2 and endostatin, FGF10, FGF-21, FGF-2, platelet derived growth factors (PDGF) including, but not limited to, PDGF-A, epidermal growth factor (EGF), vascular endothelial growth factors (VEGF) including, but not limited to, VEGF -A and VEGF-C, placenta growth factor (P1GF), pro- angiogenic growth factors and pro-lymphogenic growth factors.

[00173] In some embodiments, the active agent is a protein or peptide. Examples of protein active agents include, but are not limited to, cytokines and their receptors, as well as chimeric proteins including cytokines or their receptors, including, for example tumor necrosis factor alpha and beta, their receptors and their derivatives; renin; lipoproteins; colchicine; prolactin; corticotrophin; vasopressin; somatostatin; lypressin; pancreozymin; leuprolide; alpha- 1- antitrypsin; clotting factors such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; anti-clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator other than a tissue-type plasminogen activator (t-PA), for example a urokinase; bombesin; thrombin; hemopoietic growth factor; enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein (MIP-1 -alpha); a serum albumin such as human serum albumin; mullerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; chorionic gonadotropin; a microbial protein, such as beta-lactamase; DNase; inhibin; activin; receptors for hormones or growth factors; integrin; protein A or D; rheumatoid factors; platelet-derived growth factor (PDGF); epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-a and TGF-0, including TGF-0I, TGF-2, TGF-3, TGF-4, or TGF-5; insulin-like growth factor-I and -II (IGF-I and IGF-II); des(l-3)- IGF-I (brain IGF-I), insulin-like growth factor binding proteins; CD proteins such as CD- 3, CD-4, CD-8, and CD- 19; erythropoietin; osteoinductive factors; immunotoxins; an interferon such as interferon-alpha (e g., interferon alpha 2A), -beta, -gamma, - lambda and consensus interferon; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factor; transport proteins; homing receptors; addressins; fertility inhibitors such as the prostaglandins; fertility promoters; regulatory proteins; antibodies (including fragments thereof) and chimeric proteins, such as immunoadhesins; precursors, derivatives, prodrugs and analogues of these compounds, and pharmaceutically acceptable salts of these compounds, or their precursors, derivatives, prodrugs and analogues. Suitable proteins or peptides may be native or recombinant and include, e.g., fusion proteins.

[00174] Examples of protein active agents also include CCL1, CCL2 (MCP-1), CCL3 (MIP-Ia), CCL4 (MIP-ip), CCL5 (RANTES), CCL6, CCL7, CCL8, CCL9 (CCL10), CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1 (KC), CXCL2 (SDFla), CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8 (IL8), CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CX3CL1, XCL1, XCL2, TNFA, TNFB (LTA), TNFC (LTB), TNFSF4, TNFSF5 (CD40LG), TNFSF6, TNFSF7, TNFSF8, TNFSF9, TNFSF10, TNFSF11, TNFSF13B, EDA, IL2, IL15, IL4, IL13, IL7, IL9, IL21, IL3, IL5, IL6, IL1 1, IL27, IL30, IL31, OSM, LIF, CNTF, CTF1, IL12a, IL12b, IL23, IL27, IL35, IL14, IL16, IL32, IL34, IL10, IL22, IL19, IL20, IL24, IL26, IL29, IFNL1, IFNL2, IFNL3, IL28, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA21, IFNB1, IFNK, IFNW1, IFNG, ILIA (IL1F1), IL1B (IL1F2), ILIRa (IL1F3), IL1F5 (IL36RN), IL1F6 (IL36A), IL1F7 (IL37), IL1F8 (IL36B), IL1F9 (IL36G), ILIFIO (IL38), IL33 (IL1F11), IL18 (IL1G), IL17, KITLG, IL25 (IL17E), CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (G-CSF), SPP1, TGFB1, TGFB2, TGFB3, CCL3L1, CCL3L2, CCL3L3, CCL4L1, CCL4L2, IL17B, IL17C, IL17D, IL17F, AIMP1 (SCYE1), MIF, Areg, BC096441, Bmpl, BmplO, Bmpl5, Bmp2, Bmp3, Bmp4, Bmp5, Bmp6, Bmp7, Bmp8a, Bmp8b, Clqtnf4, Ccl21a, Ccl27a, Cd70, Cerl, Cklf, Clcfl, Cmtm2a, Cmtm2b, Cmtm3, Cmtm4, Cmtm5, Cmtm6, Cmtm7, Cmtm8, Crlfl, Ctf2, Ebi3, Ednl, Fam3b, Fasl, Fgf2, Flt31, GdflO, Gdfl 1, Gdfl5, Gdf2, GdfB, Gdf5, Gdf6, Gdf7, Gdf9, Gml2597, Gml3271, Gml3275, Gml3276, Gml3280, Gml3283, Gm2564, Gpil, Greml, Grem2, Grn, Hmgbl, Ifnal 1, Ifnal2, Ima9, Ifnab, Ifne, 1117a, 1123a, 1125, 1131, Iltifb,Inhba, Leftyl, Lefty 2, Mstn, Nampt, Ndp, Nodal, Pf4, Pgly 1, Prl7dl, Scg2, Scgb3al, Slurpl, Sppl, Thpo, TnfsflO, Tnfsfl 1, Tnfsfl2, Tnfsfl3, Tnfsfl3b, Tnfsfl4, Tnfsfl5, Tnfsfl8, Tnfsf4, Tnfsf8, Tnfsf9, Tslp, Vegfa, Wntl, Wnt2, Wnt5a, Wnt7a, Xcll, Epinephrine, Melatonin, Triiodothyronine, Thyroxine, Prostaglandins, Leukotrienes, Prostacyclin, Thromboxane, Islet Amyloid Polypeptide, Miillerian inhibiting factor or hormone, Adiponectin, Corticotropin, Angiotensin, vasopressin, arginine vasopressin, atriopeptin, Brain natriuretic peptide, Calcitonin, Cholecystokinin, Cortistatin, Enkephalin, Endothelin, Erythropoietin, Follicle-stimulating hormone, Galanin, Gastric inhibitory polypeptide, Gastrin, Ghrelin, Glucagon, Glucagon-like peptide- 1, Gonadotropin- releasing hormone, Growth hormone-releasing hormone, Hepcidin, Human chorionic gonadotropin, Human placental lactogen, Growth hormone, Inhibin, Insulin, Somatomedin, Leptin, Lipotropin, Luteinizing hormone, Melanocyte stimulating hormone, Motilin, Orexin, Oxytocin, Pancreatic polypeptide, Parathyroid hormone, Pituitary adenylate cyclase-activating peptide, Prolactin, Prolactin releasing hormone, Relaxin, Renin, Secretin, Somatostatin, Thrombopoietin, Thyrotropin, Thyrotropinreleasing hormone, Vasoactive intestinal peptide, Androgen, Androgen, acid maltase (alphaglucosidase), glycogen phosphorylase, glycogen debrancher enzyme, Phosphofructokinase, Phosphogly cerate kinase, Phosphogly cerate mutase, Lactate dehydrogenase, Carnitine palymityl transferase, Carnitine, and Myoadenylate deaminase.

[00175] Hormones to be included in the disclosed devices or to be produced from cells included in the devices can be any hormone of interest. Examples of endocrine hormones include Anti-diuretic Hormone (ADH), which is produced by the posterior pituitary, targets the kidneys, and affects water balance and blood pressure; Oxytocin, which is produced by the posterior pituitary, targets the uterus, breasts, and stimulates uterine contractions and milk secretion; Growth Hormone (GH), which is produced by the anterior pituitary, targets the body cells, bones, muscles, and affects growth and development; Prolactin, which is produced by the anterior pituitary, targets the breasts, and maintains milk secretions; Growth Hormone-Releasing Hormone (GHRH), which is a releasing hormone of GH and is produced in the arcuate nucleas of the hypothalamus; Thyroid Stimulating Hormone (TSH), which is produced by the anterior pituitary, targets the thyroid, and regulates thyroid hormones; Thyrotropin- Release Hormone (TRH), which is produced by the hypothalamus and stimulates the release of TSH and prolactin from the anterior pituitary; Adrenocorticotropic Hormone (ACTH), which is produced by the anterior pituitary, targets the adrenal cortex, and regulates adrenal cortex hormones; Follicle-Stimulating Hormone (FSH), which is produced by the anterior pituitary, targets the ovaries/testes, and stimulates egg and sperm production; Lutenizing Hormone (LH), which is produced by the anterior pituitary, targets the ovaries/testes, and stimulates ovulation and sex hormone release; Luteinizing Hormone-Releasing Hormone (LHRH), also known as Gonadotropin- Releasing Hormone (GnRH), which is synthesized and released from GnRH neurons within the hypothalamus and is a trophic peptide hormone responsible for the release of FSH and LH; Thyroxine, which is produced by the thyroid, targets the body cells, and regulates metabolism; Calcitonin, which is produced by the thyroid, targets the adrenal cortex, and lowers blood calcium; Parathyroid Hormone, which is produced by the parathyroid, targets the bone matrix, and raises blood calcium; Aldosterone, which is produced by the adrenal cortex, targets the kidney, and regulates water balance; Cortisol, which is produced by the adrenal cortex, targets the body cells, and weakens immune system and stress responses; Epinephrine, which is produced by the adrenal medulla, targets the heart, lungs, liver, and body cells, and affects primary "fight or flight" responses; Glucagon, which is produced by the pancreas, targets the liver body, and raises blood glucose level; Insulin, which is produced by the pancreas, targets body cells, and lowers blood glucose level; Estrogen, which is produced by the ovaries, targets the reproductive system, and affects puberty, menstrual, and development of gonads; Progesterone, which is produced by the ovaries, targets the reproductive system, and affects puberty, menstrual cycle, and development of gonads; and Testosterone, which is produced by the adrenal gland, testes, targets the reproductive system, and affects puberty, development of gonads, and sperm.

[00176] In some embodiments, the protein is a growth hormone, such as human growth hormone (hGH), recombinant human growth hormone (rhGH), bovine growth hormone, methione-human growth hormone, des-phenylalanine human growth hormone, and porcine growth hormone; insulin, insulin A-chain, insulin B-chain, and proinsulin; or a growth factor, such as vascular endothelial growth factor (VEGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), transforming growth factor (TGF), and insulin-like growth factor-I and -II (IGF-I and IGF-II).

[00177] Biologically active polynucleotides

[00178] Further to the bioactive agents outlined above, compositions of the present disclosure may comprise biologically active polynucleotides. The biologically active polynucleotides may, for example, encode any of the bioactive agents listed in the previous section. In some cases, these can comprise single stranded or double stranded RNA or DNA. It should be clear that the present disclosure is not limited to the specific nucleic acids disclosed herein. The present disclosure is not limited in scope to any particular source, sequence, or type of nucleic acid, however, as one of ordinary skill in the art could readily identify related homologs in various other sources of the nucleic acid including nucleic acids from non-human species (e.g., mouse, rat, rabbit, dog, monkey, gibbon, chimp, ape, baboon, cow, pig, horse, sheep, cat and other species). It is contemplated that the nucleic acid used in the present disclosure can comprise a sequence based upon a naturally-occurring sequence.

[00179] The amount of nucleic acid encapsulated by or located within the lipid nanoparticle may vary based on the intended use. The amount of nucleic acid may be calculated as a ratio with respect to the lipid nanoparticle composition (w/w) or to any of the individual components of the lipid nanoparticle composition (w/w). For example, the ratio of cationic ionizable lipid to nucleic acid may be about from about 50: 1 (w/w), about 20: 1 (w/w), about 15:1 (w/w), about 14: 1 (w/w), about 13: 1 (w/w), about 12: 1 (w/w), about 11 : 1 (w/w), about 10: 1 (w/w), about 9: 1 (w/w), about 8: 1 (w/w), about 7:1 (w/w), about 6:1 (w/w), to about 5:1 (w/w), or any range derivable therein. In some embodiments, the ratio of cationic ionizable lipid to nucleic acid is about 11.33 (w/w). The length of the nucleic acid encapsulated by or located within the lipid nanoparticle may also vary based on the intended use. The length of the nucleic acid may be about 20 bp, about 50 bp, about 75 bp, about 100 bp, about 150 bp, about 200 bp, about 250 bp, about 300 bp, about 350 bp, about 400 bp, about 450 bp, about 500 bp, about 550 bp, about 600 bp, about 650 bp, about 700 bp, about 750 bp, about 800 bp, about 850 bp, about 900 bp, about 950 bp, about 1000 bp, or any range derivable therein. Longer nucleic acids are also contemplated, such as nucleic acids that are about 1500 bp, about 2000 bp, about 2500 bp, about 3000 bp, about 3500 bp, about 4000 bp, about 4500 bp, about 5000 bp, about 5500 bp, about 6000 bp, about 6500 bp, about 7000 bp, about 7500 bp, about 8000 bp, about 8500 bp, about 9000 bp, about 9500 bp, about 10,000 bp, or any range derivable therein.

[00180] In some aspects, the nucleic acid is a sequence which silences, is complimentary to, or replaces another sequence present in vivo. Sequences of 17 bases in length should occur only once in the human genome and, therefore, suffice to specify a unique target sequence. Although shorter oligomers are easier to make and increase in vivo accessibility, numerous other factors are involved in determining the specificity of hybridization. Both binding affinity and sequence specificity of an oligonucleotide to its complementary target increases with increasing length. It is contemplated that exemplary oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more base pairs will be used, although others are contemplated. Longer polynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 or longer are contemplated as well.

[00181] The nucleic acid used herein may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism. In preferred embodiments, however, the nucleic acid would comprise complementary DNA (cDNA). Also contemplated is a cDNA plus a natural intron or an intron derived from another gene; such engineered molecules are sometime referred to as "mini -genes." At a minimum, these and other nucleic acids of the present disclosure may be used as molecular weight standards in, for example, gel electrophoresis.

[00182] The term "cDNA" is intended to refer to DNA prepared using messenger RNA (mRNA) as template. The advantage of using a cDNA, as opposed to genomic DNA or DNA polymerized from a genomic, non- or partially-processed RNA template, is that the cDNA primarily contains coding sequences of the corresponding protein. There may be times when the full or partial genomic sequence is preferred, such as where the non-coding regions are required for optimal expression or where non-coding regions such as introns are to be targeted in an antisense strategy.

[00183] In some embodiments, the nucleic acid comprises one or more antisense segments which inhibits expression of a gene or gene product. Antisense methodology takes advantage of the fact that nucleic acids tend to pair with "complementary" sequences. By complementary, it is meant that polynucleotides are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Inclusion of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.

[00184] Targeting double-stranded (ds) DNA with polynucleotides leads to triple-helix formation; targeting RNA will lead to double-helix formation. Antisense polynucleotides, when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability. Antisense RNA constructs, or DNA encoding such antisense RNA's, may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.

[00185] Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. It is contemplated that the most effective antisense constructs will include regions complementary to intron/exon splice junctions. Thus, it is proposed that a preferred embodiment includes an antisense construct with complementarity to regions within 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected.

[00186] As stated above, "complementary" or "antisense" means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated. For example, an antisense construct which has limited regions of high homology, but also contains a non-homologous region (e.g., ribozyme; see below) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.

[00187] It may be advantageous to combine portions of genomic DNA with cDNA or synthetic sequences to form a siRNA or to generate specific constructs. For example, where an intron is desired in the ultimate construct, a genomic clone will need to be used. The cDNA, siRNA, or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence. Other embodiments include dsRNA or ssRNA, which may be used to target genomic sequences or coding/non-coding transcripts.

[00188] In other embodiments, the compositions may comprise a nucleic acid which comprises one or more expression vectors are used in a gene therapy. Expression requires that appropriate signals be provided in the vectors, and which include various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells. Elements designed to optimize messenger RNA stability and translatability in host cells also are defined. The conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products are also provided, as is an element that links expression of the drug selection markers to expression of the polypeptide.

[00189] Throughout this application, the term "expression construct" is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed. The transcript may be translated into a protein, but it need not be. In certain embodiments, expression includes both transcription of a gene and translation of mRNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding a gene of interest.

[00190] The term "vector" is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. A nucleic acid sequence can be "exogenous," which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques, which are described in Sambrook et al. (1989) and Ausubel et al. (1994), both incorporated herein by reference.

[00191] The term "expression vector" refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra. mRNA

[00192] In some aspects, the present compounds and compositions may be used in the delivery of an mRNA to a cell. Messenger RNA or mRNA are short RNA strands which transfer the genetic code from the DNA to the ribosomes so the mRNA may be translated into a therapeutic protein or peptide, or an antigen. The mRNAs described herein may be unprocessed or have undergone processing to add a poly(A) tail, be edited in vivo, or have a 5' cap added. The mRNA molecules may comprise a 5’ UTR or a 3’UTR. The present compositions are contemplated in the delivery of a variety of different mRNA including those which have not undergone processing or have been further processed. Additionally, these nucleic acids may be used therapeutically, used to produce an antibody in vivo, or in a vaccine formulation. mRNA molecules can provide a more direct method of expressing a polypeptide of interest in a target cell. However, such molecules are typically highly liable and rapidly degraded. In some aspects, LNP processing according to the embodiments can be used to substantially stabilize mRNA. In preferred aspects, mRNA is provided encapsulated in or in complex with LNPs.

[00193] As mentioned above, in some aspects a nucleic acid molecule of the embodiments encodes a therapeutic polypeptide. For example, the therapeutic protein may be a protein, such as an enzyme that is non-functional or disrupted in a particular disease state (e.g., CFTR in cystic fibrosis).

[00194] In further aspects, a polynucleotide of the embodiments encodes an antigen, such as an antigen from a pathogen or a cancer cell-associated antigen. For example, the cancer associated antigen can be CD 19, CD20, R0R1, CD22, carcinoembryonic antigen, alphafetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate-specific antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2/Neu, folate binding protein, GD2, CD123, CD33, CD138, CD23, CD30 , CD56, c-Met, mesothelin, GD3, HERV-K, IL-l lRalpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII or VEGFR2. In some specific aspects the antigen is GP240, 5T4, HER1, CD-33, CD-38, VEGFR-1, VEGFR-2, CEA, FGFR3, IGFBP2, IGF-1R, BAFF-R, TACI, APRIL, Fnl4, ERBB2 or ERBB3

[00195] Antigens useful in the present disclosure may include those derived from viruses including, but not limited to, those from the family Arenaviridae (e.g., Lymphocytic choriomeningitis virus), Arterivirus (e.g., Equine arteritis virus), Astroviridae (Human astrovirus 1), Birnaviridae (e.g., Infectious pancreatic necrosis virus, Infectious bursal disease virus), Bunyaviridae (e.g., California encephalitis virus Group), Caliciviridae (e g., Caliciviruses), Coronaviridae (e.g., Human coronaviruses 299E and OC43), Deltavirus (e.g., Hepatitis delta virus), Filoviridae (e.g., Marburg virus, Ebola virus), Flaviviridae (e.g., Yellow fever virus group, Hepatitis C virus), Hepadnaviridae (e.g., Hepatitis B virus), Herpesviridae (e.g., Epstein-Bar virus, Simplexvirus, Varicellovirus, Cytomegalovirus, Roseolovirus, Lymphocryptovirus, Rhadinovirus), Orthomyxoviridae (e.g., Influenzavirus A, B, and C), Papovaviridae (e.g., Papillomavirus), Paramyxoviridae (e.g., Paramyxovirus such as human parainfluenza virus 1, Morbillivirus such as Measles virus, Rubulavirus such as Mumps virus, Pneumovirus such as Human respiratory syncytial virus), Picomaviridae (e.g., Rhinovirus such as Human rhinovirus 1A, Hepatovirus such Human hepatitis A virus, Human poliovirus, Cardiovirus such as Encephalomyocarditis virus, Aphthovirus such as Foot-and-mouth disease virus O, Coxsackie virus), Poxyiridae (e.g., Orthopoxvirus such as Variola virus or monkey poxvirus), Reoviridae (e.g., Rotavirus such as Groups A-F rotaviruses), Retroviridae (Primate lentivirus group such as human immunodeficiency virus 1 and 2), Rhabdoviridae (e.g., rabies virus), Togaviridae (e.g., Rubivirus such as Rubella virus), Human T-cell leukemia virus, Murine leukemia virus, Vesicular stomatitis virus, Wart virus, Blue tongue virus, Sendai virus, Feline leukemia virus, Simian virus 40, Mouse mammary tumor virus, Dengue virus, HIV-1 and HIV-2, West Nile, H1N1, SARS, 1918 Influenza, Tick-borne encephalitis virus complex (Absettarov, Hanzalova, Hypr), Russian Spring-Summer encephalitis virus, Congo-Crimean Hemorrhagic Fever virus, Junin Virus, Kumlinge Virus, Marburg Virus, Machupo Virus, Kyasanur Forest Disease Virus, Lassa Virus, Omsk Hemorrhagic Fever Virus, FIV, SIV, Herpes simplex 1 and 2, Herpes Zoster, Human parvovirus (Bl 9), Respiratory syncytial virus, Pox viruses (all types and serotypes), Colti virus, Reoviruses — all types, and/or Rubivirus (rubella).

[00196] Antigens useful in the present disclosure may include those derived from bacteria including, but not limited to, Streptococcus agalactiae, Legionella pneumophilia, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhosae, Neisseria meningitidis, Pneumococcus, Hemophilis influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, Mycobacterium tuberculosis, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiensei, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japanicum, Babesia bovis, Elmeria tenella, Onchocerca volvulus, Leishmania tropica, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium, M. pneumoniae, Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Aspergillus fumigatus, Penicillium marneffei, Bacillus anthracis, Bartonella, Bordetella pertussis, Brucella — all serotypes, Chlamydia trachomatis, Chlamydia pneumoniae, Clostridium botulinum — anything from clostridium serotypes, Haemophilus influenzae, Helicobacter pylori, Klebsiella — all serotypes, Legionella — all serotypes, Listeria, Mycobacterium — all serotypes, Mycoplasma — human and animal serotypes, Rickettsia — all serotypes, Shigella — all serotypes, Staphylococcus aureus, Streptococcus — S. pneumoniae, S. pyogenes, Vibrio cholera, Yersinia enterocolitica, and/or Yersinia pestis.

[00197] Antigens useful in the present disclosure may include those derived from parasites including, but not limited to, Ancylostomahuman hookworms, Leishmania — all strains, Microsporidium, Necator human hookworms, Onchocerca filarial worms, Plasmodium — all human strains and simian species, Toxoplasma — all strains, Trypanosoma — all serotypes, and/or Wuchereria bancrofti filarial worms. siRNA

[00198] As mentioned above, the present disclosure contemplates the use of one or more inhibitory nucleic acid for reducing expression and/or activation of a gene or gene product. Examples of an inhibitory nucleic acid include but are not limited to molecules targeted to an nucleic acid sequence, such as an siRNA (small interfering RNA), short hairpin RNA (shRNA), double-stranded RNA, an antisense oligonucleotide, a ribozyme and molecules targeted to a gene or gene product such as an aptamer.

[00199] An inhibitory nucleic acid may inhibit the transcription of a gene or prevent the translation of the gene transcript in a cell. An inhibitory nucleic acid may be from 16 to 1000 nucleotides long, and in certain embodiments from 18 to 100 nucleotides long.

[00200] Inhibitory nucleic acids are well known in the art. For example, siRNA, shRNA and double-stranded RNA have been described in U.S. Patents 6,506,559 and 6,573,099, as well as in U.S. Patent Publications 2003/0051263, 2003/0055020, 2004/0265839, 2002/0168707, 2003/0159161, and 2004/0064842, all of which are herein incorporated by reference in their entirety.

[002011 Since the discovery of RNAi by Fire and colleagues in 1998, the biochemical mechanisms have been rapidly characterized. Double stranded RNA (dsRNA) is cleaved by Dicer, which is an RNAase III family ribonuclease. This process yields siRNAs of ~21 nucleotides in length. These siRNAs are incorporated into a multiprotein RNA-induced silencing complex (RISC) that is guided to target mRNA. RISC cleaves the target mRNA in the middle of the complementary region. In mammalian cells, the related microRNAs (miRNAs) are found that are short RNA fragments (~22 nucleotides). miRNAs are generated after Dicer-mediated cleavage of longer (~70 nucleotide) precursors with imperfect hairpin RNA structures. The miRNA is incorporated into a miRNA-protein complex (miRNP), which leads to translational repression of target mRNA.

[00202] In designing a nucleic acid capable of generating an RNAi effect, there are several factors that need to be considered such as the nature of the siRNA, the durability of the silencing effect, and the choice of delivery system. To produce an RNAi effect, the siRNA that is introduced into the organism will typically contain exonic sequences. Furthermore, the RNAi process is homology dependent, so the sequences must be carefully selected so as to maximize gene specificity, while minimizing the possibility of cross-interference between homologous, but not gene-specific sequences. Particularly the siRNA exhibits greater than 80, 85, 90, 95, 98% or even 100% identity between the sequence of the siRNA and a portion of a EphA nucleotide sequence. Sequences less than about 80% identical to the target gene are substantially less effective. Thus, the greater identity between the siRNA and the gene to be inhibited, the less likely expression of unrelated genes will be affected.

[00203] In addition, the size of the siRNA is an important consideration. In some embodiments, the present disclosure relates to siRNA molecules that include at least about 19-25 nucleotides, and are able to modulate gene expression. In the context of the present disclosure, the siRNA is particularly less than 500, 200, 100, 50, 25, or 20 nucleotides in length. In some embodiments, the siRNA is from about 25 nucleotides to about 35 nucleotides or from about 19 nucleotides to about 25 nucleotides in length.

[00204] To improve the effectiveness of siRNA-mediated gene silencing, guidelines for selection of target sites on mRNA have been developed for optimal design of siRNA (Soutschek et al., 2004; Wadhwa et al., 2004). These strategies may allow for rational approaches for selecting siRNA sequences to achieve maximal gene knockdown. To facilitate the entry of siRNA into cells and tissues, a variety of vectors including plasmids and viral vectors such as adenovirus, lentivirus, and retrovirus have been used (Wadhwa et al., 2004).

[00205] Within an inhibitory nucleic acid, the components of a nucleic acid need not be of the same type or homogenous throughout (e.g., an inhibitory nucleic acid may comprise a nucleotide and a nucleic acid or nucleotide analog). Typically, an inhibitory nucleic acid form a double-stranded structure; the double-stranded structure may result from two separate nucleic acids that are partially or completely complementary. In certain embodiments of the present disclosure, the inhibitory nucleic acid may comprise only a single nucleic acid (polynucleotide) or nucleic acid analog and form a double-stranded structure by complementing with itself (e.g., forming a hairpin loop). The double-stranded structure of the inhibitory nucleic acid may comprise 16-500 or more contiguous nucleobases, including all ranges derivable thereof. The inhibitory nucleic acid may comprise 17 to 35 contiguous nucleobases, more particularly 18 to 30 contiguous nucleobases, more particularly 19 to 25 nucleobases, more particularly 20 to 23 contiguous nucleobases, or 20 to 22 contiguous nucleobases, or 21 contiguous nucleobases that hybridize with a complementary nucleic acid (which may be another part of the same nucleic acid or a separate complementary nucleic acid) to form a double-stranded structure.

[00206] siRNA can be obtained from commercial sources, natural sources, or can be synthesized using any of a number of techniques well-known to those of ordinary skill in the art. For example, commercial sources of predesigned siRNA include Invitrogen’s StealthTM Select technology (Carlsbad, CA), Ambion®(Austin, TX), and Qiagen® (Valencia, CA). An inhibitory nucleic acid that can be applied in the compositions and methods of the present disclosure may be any nucleic acid sequence that has been found by any source to be a validated downregulator of the gene or gene product.

[00207] In some embodiments, the disclosure features an isolated siRNA molecule of at least 19 nucleotides, having at least one strand that is substantially complementary to at least ten but no more than thirty consecutive nucleotides of a nucleic acid that encodes a gene, and that reduces the expression of a gene or gene product. In one embodiments of the present disclosure, the siRNA molecule has at least one strand that is substantially complementary to at least ten but no more than thirty consecutive nucleotides of the mRNA that encodes a gene or a gene product. [00208] In one embodiments, the siRNA molecule is at least 75, 80, 85, or 90% homologous, particularly at least 95%, 99%, or 100% similar or identical, or any percentages in between the foregoing (e.g., the disclosure contemplates 75% and greater, 80% and greater, 85% and greater, and so on, and said ranges are intended to include all whole numbers in between), to at least 10 contiguous nucleotides of any of the nucleic acid sequences encoding a target therapeutic protein.

[00209] The siRNA may also comprise an alteration of one or more nucleotides. Such alterations can include the addition of non-nucleotide material, such as to the end(s) of the 19 to 25 nucleotide RNA or internally (at one or more nucleotides of the RNA). In certain aspects, the RNA molecule contains a 3'-hydroxyl group. Nucleotides in the RNA molecules of the present disclosure can also comprise non-standard nucleotides, including non-naturally occurring nucleotides or deoxyribonucleotides. The double-stranded oligonucleotide may contain a modified backbone, for example, phosphorothioate, phosphorodi thioate, or other modified backbones known in the art, or may contain non-natural internucleoside linkages. Additional modifications of siRNAs (e.g., 2'-O-methyl ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, “universal base” nucleotides, 5-C-methyl nucleotides, one or more phosphorothioate internucleotide linkages, and inverted deoxyabasic residue incorporation) can be found in U.S. Publication 2004/0019001 and U.S. Patent 6,673,611 (each of which is incorporated by reference in its entirety). Collectively, all such altered nucleic acids or RNAs described above are referred to as modified siRNAs.

[00210] In one embodiment, siRNA is capable of decreasing the expression of a particular genetic product by at least 10%, at least 20%, at least 30%, or at least 40%, at least 50%, at least 60%, or at least 70%, at least 75%, at least 80%, at least 90%, at least 95% or more or any ranges in between the foregoing.

[00211] Features of Implantable Compositions

[00212] In some embodiments, the present tissue mimicking compositions may be implanted in a subject. In some embodiments, the compositions described herein may be used for the treatment or prevention of a disease or disorder in a patient in need thereof. The present compositions comprise one or more channels, which also have features described in the sections that follow. The implantable composition or the component parts (for example, the channels or the biomaterial) described herein may take any suitable shape or morphology. For example, an implantable composition may be a sphere, spheroid, tube, cord, string, ellipsoid, disk, cylinder, sheet, torus, cube, stadiumoid, cone, pyramid, triangle, rectangle, square, or rod. The channels of the present compositions may be a sphere, spheroid, tube, cord, string, ellipsoid, disk, cylinder, sheet, torus, cube, stadiumoid, cone, pyramid, triangle, rectangle, square, rod, or any combination thereof. An implantable composition may comprise a curved or flat section. The channels of the present compositions may comprise a curved or flat section. The biomaterial may comprise a curved or a flat section. In an embodiment, an implantable composition may be prepared through the use of a mold, resulting in a custom shape. In an embodiment, an implantable composition may be prepared through the use of a pattern, resulting in a custom shape. In some embodiments, the custom shape may resemble a naturally-occuring feature, such as an organ. In some embodiments, the custom shape may resemble a lymph node.

[00213] The implantable composition as disclosed herein may vary in size, depending, for example, on the use or site of implantation. For example, an implantable composition may have a mean diameter or size greater than 0.1 mm, e.g., greater than 0.25 mm, 0.5 mm, 0.75, 1 mm, 2 mm, 4 mm, 6 mm, 8 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 30 mm, 40 mm, 50 mm, or more. In an embodiment, an implantable composition may have a section or region with a mean diameter or size greater than 0.1 mm, e.g., greater than 0.25 mm, 0.5 mm, 0.75, 1 mm, 1.5 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 20 mm, 30 mm, 40 mm, 50 mm, or more. In an embodiment, an implantable composition may have a mean diameter or size less than 20 mm, e.g., less than 20 mm, 19 mm, 18 mm, 17 mm, 16 mm, 15 mm, 14 mm, 13 mm, 12 mm, 11 mm, 10 mm, 9 mm, 8 mm, 7 mm, 6 mm, 5 mm, or smaller. In an embodiment, an implantable composition may have a section or region with a mean diameter or size less than 15 mm, e.g., less than 15 mm, 12.5 mm, 10 mm, 9 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4 mm, 3 mm, 2 mm, 1 mm, 0.5 mm, or smaller.

[00214] In an embodiment, an implantable composition comprises a pore or opening to permit passage of an object, such as a small molecule (e.g., nutrients or waste), a protein, or a nucleic acid. Such a pore may be located with the composition (that is, connecting regions, areas, or features within the composition). For example, a pore in or on an implantable composition may be greater than 0.1 nm and less than 10 pm. In an embodiment, the implantable composition comprises a pore or opening with a size range of 0.1 pm to 10 pm, 0.1 pm to 9 pm, 0.1 pm to 8 pm, 0.1 gm to 7 pm, 0.1 pm to 6 pm, 0.1 pm to 5 pm, 0.1 pm to 4 pm, 0.1 pm to 3 pm, 0.1 pm to 2 pm.

[00215] An implantable composition described herein may comprise a chemical modification in or on any enclosed material. Exemplary chemical modifications include small molecules, peptides, proteins, nucleic acids, lipids, or oligosaccharides, examples of which are provided in a previous section. The implantable composition may comprise at least 0.5%, 1%, 2%, 3%, 4%, 5%, 7.5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of a material that is chemically modified, e.g., with a chemical modification described herein. An implantable composition may be partially coated with a chemical modification, e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.9% coated with a chemical modification.

[00216] In an embodiment, the implantable composition is formulated such that the duration of release of the antigenic and/or therapeutic agent is tunable. For example, an implantable composition may be configured in a certain manner to release a specific amount of an antigenic or therapeutic agent over time, e.g., in a sustained or controlled manner. In an embodiment, the implantable composition comprises a material that is semi-permeable, and this material affects the duration of therapeutic release from the construct by gradually ceasing immunoprotection of encapsulated cells or causing gradual release of the antigenic agent.

[00217] In some embodiments, the implantable composition comprises a zone that is targeted by the natural foreign body response (FBR) of a host or subject, e.g., over a period of time. In an embodiment, the implantable composition is coated with fibrotic overgrowth upon administration to a subject, e.g., over a period of time. Fibrotic overgrowth on the surface of the implantable composition may lead to a decrease in function of the implantable composition. For example, a decrease in function may comprise a reduction in the release of an antigenic or therapeutic agent over time, a decrease in pore size, or a decrease in the diffusion rate of oxygen and other key nutrients to the encapsulated cells, leading to cell death. In an embodiment, the rate of fibrotic overgrowth may be tuned to design a dosing regimen. For example, the fibrotic overgrowth on the surface of an implantable composition may result in a decrease in function of the implantable composition about 6 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 2.5 weeks, 3 weeks, 4 weeks, or 6 weeks after administration (e.g., injection or implantation) to a subject. [00218] In some embodiments, the implantable composition is chemically modified with a specific density of modifications. The specific density of chemical modifications may be described as the average number of attached chemical modifications per given area. For example, the density of a chemical modification on or in an implantable composition may be 0.01, 0.1, 0.5, 1, 5, 10, 15, 20, 50, 75, 100, 200, 400, 500, 750, 1,000, 2,500, or 5,000 chemical modifications per square pm or square mm.

[00219] An implantable composition may be formulated or configured for implantation in any organ, tissue, cell, or part of a subject. For example, the implantable composition may be implanted or disposed into the intraperitoneal space of a subject. An implantable composition may be implanted in or disposed on a tumor or other growth in a subject, or be implanted in or disposed about 0.1 mm, 0.5 mm, 1 mm, 0.25 mm, 0.5 mm, 0.75, 1 mm, 1.5 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 20 mm, 30 mm, 40 mm, 50 mm, 1 cm, 5, cm, 10 cm, or further from a tumor or other growth in a subject. An implantable composition may be configured for implantation, or implanted, or disposed on or in the skin, a mucosal surface, a body cavity, the central nervous system (e.g., the brain or spinal cord), an organ (e.g., the heart, eye, liver, kidney, spleen, lung, ovary, breast, uterus), the lymphatic system, vasculature, oral cavity, nasal cavity, gastrointestinal tract, bone, muscle, adipose tissue, skin, or other area.

[00220] An implantable composition may be formulated for use for any period of time. For example, an implantable composition may be used for 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or longer. An implantable composition can be configured for limited exposure (e.g., less than 2 days, e.g., less than 2 days, 1 day, 24 hours, 20 hours, 16 hours, 12 hours, 10 hours, 8 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour or less). A implantable composition can be configured for prolonged exposure (e.g., at least 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years or more). An implantable composition can be configured for permanent exposure (e.g., at least 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years or more).

III. Pro-lymphangiogenic and pro-angiogenic cell engineering platforms

[00221] Provided herein are pro-lymphangiogenic and pro-angiogenic cell engineering platforms. In some embodiments, the platform includes engineered tissue mimicking compositions, wherein said compositions comprise a hydrogel polymer and/or engineered cells.

[00222] The cell engineered platforms described herein can be used for long-term disease management, drug delivery, engineered cell therapy, vascular, vascularizing materials/therapy, and/or regenerative material/therapy. These platforms can also be used to facilitate lymphatic regeneration and/or vascular regeneration, and for guiding lymphatic and/or vascular growth. Other uses for these platforms include production of lymphatic and blood regenerative molecules and regeneration/growth/maintenance/maturation of lymphatic and blood vessels for but not limited to therapeutics for disease management. For example, embodiments can comprise vascularized hydrogel platforms that include but are not limited to tissue grafts and implants for the treatment of various diseases such as autoimmune disorders, metabolic diseases, oncology, neurology, inflammation, cardiovascular disease, and others.

[00223] In some embodiments, the cell engineered platform can produce and secrete of any of the therapeutic agents, proteins or nucleic acids (e.g., a therapeutic protein or nucleic acid) described herein. In some embodiments, the cell engineered platform can produce and/or secrete any of the pro-angiogenic and pro-lymphogenic growth factors described herein including, but not limited to, Epidermal Growth Factor (EGF), Platelet Derived Growth Factor A (PDGF-A), Vascular Endothelial Growth Factor A (VEGF-A), Vascular Endothelial Growth Factor C (VEGF- C), Placental Growth Factor (Pl GF), Fibroblast Growth Factors 2, 10 and 21 (FGF2, FGF10 and FGF21), or combinations thereof.

[00224] In some embodiments, the engineered cells included in the cell engineered platform can be any of the engineered cells described herein. In some embodiments, the engineered cell included in the cell engineered platform can be any cell derived from a human or a non-human mammal. In some embodiments, the engineered cell included in the cell engineered platform can be a cell from the retina. In some embodiments, the engineered cell can be a pigment epithelial cell. In some embodiments, the engineered cell is an Adult Retinal Pigment Epithelial cell line-19 (ARPE-19). In some embodiments the cell engineered platform includes genetically engineered ARPE-19 cells.

[00225] In some embodiments any of the cells described herein (e.g., ARPE-19) can be genetically modified to insert a desired DNA using any suitable method described in the art. In some embodiments any of the cells described herein (e g., ARPE-19) can be genetically modified using a transposon-based system including, but not limited to, piggyBac® and Sleeping Beauty, for the insertion of a DNA fragment encoding for any of the therapeutic agents described herein (e g., pro-angiogenic and pro-lymphogenic growth factors) including, but not limited to, Epidermal Growth Factor (EGF), Platelet Derived Growth Factor A (PDGF-A), Vascular Endothelial Growth Factor A (VEGF-A), Vascular Endothelial Growth Factor C (VEGF-C), Placental Growth Factor (P1GF), and/or Fibroblast Growth Factors 2, 10 and 21 (FGF2, FGF10 and FGF21).

[00226] The piggy Bac transposon is a Class 2 transposon originally isolated from Trichoplusia ni (cabbage looper moth) that has been shown to actively transpose in mammalian cells, with a preference for accessible chromatin structures (WO2023230272). Because it can introduce exogeneous DNA into a genome and promote stable transgene expression, the piggyBac transposon system can be a useful tool for genetic manipulation in mammalian cells and can be used to facilitate the stable transfection of mammalian cells, to generate stable and high producing polyclonal cultures of mammalian cells, to produce recombinant proteins from heterogeneous populations of transfected cells and to develop clones for cell line development (see WO2020123327). One of skill in the art would be well equipped to construct engineered cells through standard transposon-based techniques, which are described in the art and in WO2023230272, incorporated herein by reference. In some embodiments, ARPE-19 cells are genetically modified using a transposon-based system (e.g., PiggyBac®) to insert into the ARPE- 19 cells a DNA fragment encoding any of the pro-angiogenic and pro-lymphogenic growth factors described herein including, but not limited to, Epidermal Growth Factor (EGF), Platelet Derived Growth Factor A (PDGF-A), Vascular Endothelial Growth Factor A (VEGF-A), Vascular Endothelial Growth Factor C (VEGF-C), Placental Growth Factor (P1GF), and/or Fibroblast Growth Factors 2, 10 and 21 (FGF2, FGF10 and FGF21).

[00227] The piggyBac transposon has a broad host spectrum. This mobile element has been widely used for a variety of applications in a diverse range of organisms (Yusa K. 2014. piggyBac transposon. Microbiol. Spectrum 3(2):MDNA3-0028-2014). In some embodiments, the cell engineering platform described herein can include engineered cells from various species including, but not limited to, yeast, mice, mammals and humans wherein the cells can produce and/or secrete any of the therapeutic agents described herein (e.g., pro-angiogenic or pro-lymphogenic factors) including, but not limited to, Epidermal Growth Factor (EGF), Platelet Derived Growth Factor A (PDGF-A), Vascular Endothelial Growth Factor A (VEGF-A), Vascular Endothelial Growth Factor C (VEGF-C), Placental Growth Factor (P1GF), and/or Fibroblast Growth Factors 2, 10 and 21 (FGF2, FGF10 and FGF21).

[00228] In some embodiments, the amount of DNA included in the engineered cells (e.g., ARPE-19) can be controlled to express, produced and/or secrete a target amount of pro- lymphogenic and/or pro-angiogenic proteins. The piggyBac™ system allows for random integration of DNA that is achieved using the piggyBac™ transposase, which facilitates direct integration of the donor-transposon (carrying your cargo of interest) into random ‘TTAA’ sites throughout the genome. The cargo is inserted into the genome fully intact, and the number of integration copies can be controlled by titrating the transposase to transposon ratios. In some embodiments, the secretion of pro-angiogenic and pro-lymphogenic growth factors produced by the cell engineering platform described herein can be controlled (e.g., by titrating the transposase to transposon ratios). In some embodiments, high ratios of transposase to transposon result in a greater number of integrations per cell. In some embodiments, the transposase to transposon ratios can be adjusted to maximize or minimize expression levels of a protein and/or gene of interest. In some embodiments, the secretion of pro-angiogenic and pro-lymphogenic growth factors produced by the cell engineering platform described herein cannot be controlled.

[00229] In some embodiments, the engineered cell included in any of the cell engineered platforms described herein can express, produced and/or secrete an amount of therapeutic agent (e.g., any of the pro-angiogenic and pro-lymphogenic growth factors described herein) greater than 0.00001 pg/ml/day; e.g., greater than about 0.0001, 0.001, 0.01, 1, 10, 100, 200, 300, 400, 500, 1,000 pg/ml/day; or between about 0.00001 pg/ml/day and about 0.0001 pg/ml/day, 0.00001 pg/ml/day and about 0.0001 pg/ml/day, 0.00007 pg/ml/day and about 0.0015 pg/ml/day, 0.001 pg/ml/day and about 0.03 pg/ml/day, 0.01 pg/ml/day and about 0.15 pg/ml/day, 0.1 pg/ml/day and about 1.0 pg/ml/day, 0.7 pg/ml/day and about 10 pg/ml/day, 7 pg/ml/day and about 100 pg/ml/day, 80 pg/ml/day and about 170 pg/ml/day, 150 pg/ml/day and about 260 pg/ml/day, 240 pg/ml/day and about 350 pg/ml/day, 300 pg/ml/day and about 600 pg/ml/day, 400 pg/ml/day and about 700 pg/ml/day, 500 pg/ml/day and about 800 pg/ml/day. In some embodiments, the level of can be achieved by titrating the transposase to transposon ratios used to generate the engineered cells.

[00230] In some embodiments, the cell engineered platform can include one or a plurality of cells. In some embodiments, the plurality of cells can include one or more types of cells. In some embodiments, the plurality of cells can be grouped into clusters (FIG. 7A). In some embodiments, the cells in a cluster can share a common function. In some embodiments, the cell engineered platform can include one or a plurality of clusters of cells. In some embodiments, a cluster of cells expresses, secretes and/or produce any of the pro-angiogenic or pro-lymphogenic factors including, but not limited to, Epidermal Growth Factor (EGF), Platelet Derived Growth Factor A (PDGF-A), Vascular Endothelial Growth Factor A (VEGF-A), Vascular Endothelial Growth Factor C (VEGF-C), Placental Growth Factor (P1GF), and/or Fibroblast Growth Factors 2, 10 and 21 (FGF2, FGF10 and FGF21).

[00231] In some embodiments, the engineered cells can include a DNA insert encoding for any of the therapeutic agents (e.g., pro-angiogenic or pro-lymphogenic factors described herein) wherein the DNA insert can be operatively linked to, or under the control of, a promoter. In some embodiments, the promoter is operatively linked to the DNA insert by being spatially connected. In some embodiments, the promoter can be selected to obtain any of the amounts/ranges of secreted therapeutic agents (e.g., pro-angiogenic or pro-lymphogenic factors described herein). Example of promoters that can be included in the platforms described herein include, but are not limited to, late or early SV40 promoters, the Cytomegalovirus promoter (CMV) promoter (U.S. Pat. Nos. 5,168,062; 5,385,839), an HSV tk promoter, a PGK (phosphoglycerate kinase) promoter, an EF-1 alpha promoter (U.S. Pat. No.5, 266, 491), RSV, UbiC. In some embodiments, the promoter can be a strong promoter suitable for use in the PiggyBac-engineered ARPE-19 cells. In some embodiments, the promoter can be a weak promoter suitable for use in the PiggyBac-engineered ARPE-19 cells. In some embodiments, strong and weak promoters can be used to adjust amounts/ranges of secreted therapeutic agents (e.g., pro-angiogenic or pro-lymphogenic factors described herein) expressed, produced and/or secreted by the engineered cells. In some embodiments, the promoter operatively linked to the DNA insert encoding for any of the therapeutic agents (e.g., pro-angiogenic or pro-lymphogenic factors described herein) is CMV. [00232] In some embodiments, the engineered cells include at least one selectable marker. Such markers include, e.g., but are not limited to, ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418/Geneticin (neo gene), DHFR (encoding Dihydrofolate Reductase and conferring resistance to Methotrexate), mycophenolic acid, or glutamine synthetase (GS, U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739), blasticidin (bsd gene), resistance genes for eukaryotic cell culture as well as ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418/Geneticin (neo gene), kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B, or tetracycline resistance genes for culturing in E. coli and other bacteria or prokaryotics (the above patents are entirely incorporated hereby by reference). In some embodiments, the selectable marker included in the engineered cells is puromycin. In some embodiments, the engineered cells expressing, producing and/or secreting any of pro-angiogenic or pro-lymphogenic factors described herein can be selected using puromycin. [00233] Signal peptides (sometimes referred to as signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide) are short amino acid sequences that direct the linked proteins into the secretory pathway. SPs are found in the N-terminus of proteins in virtually all organisms. Signal peptides are usually 16-30 amino acids long and consist of a positively charged n-region, a hydrophobic h-region, and a c-region. The c-region contains the signal peptidase recognition site (von Heijne, 1990; 1998). In some embodiments, the engineered cells can include at least one signal peptide. In some embodiments, the signal peptide can direct the expressed therapeutic agent (e.g., any of the pro-angiogenic or pro-lymphogenic factors described herein) into the secretory pathway. One of skill in the art would be well equipped to construct engineered cells including DNA vectors including signal peptides through standard techniques, which are described in the art and in Kapp et al., Protein Transport into the Endoplasmic Reticulum, edited by Richard Zimmermann, incorporated herein by reference.

[00234] In some embodiments, the cell engineered platform can include engineered monoclonal cell lines that can express, produce and/or secrete any of the pro-angiogenic or pro- lymphogenic factors described herein, including, but not limited to, Epidermal Growth Factor (EGF), Platelet Derived Growth Factor A (PDGF-A), Vascular Endothelial Growth Factor A (VEGF-A), Vascular Endothelial Growth Factor C (VEGF-C), Placental Growth Factor (P1GF), and/or Fibroblast Growth Factors 2, 10 and 21 (FGF2, FGF10 and FGF21). One of skill in the art would be well equipped to construct engineered monoclonal cell lines using standard techniques, which are described in the art and in Milstein and Kohler, Nature. 1975 Aug 7;256(5517):495-7. In some embodiments, the monoclonal engineered cell line can increase the production of any of the therapeutic agents (e.g., pro-angiogenic or pro-lymphogenic factors) described herein. In some embodiments, the monoclonal engineered cell line can stabilize during expansion of the cell line the production of any of the therapeutic agents (e.g., pro-angiogenic or pro-lymphogenic factors) described herein.

[00235] In some embodiments, the cell engineered platform described herein can include encapsulated engineered cells (FIG. 7B). In some embodiments, the engineered cells can be encapsulated in any of the natural and/or synthetic materials described herein. In some embodiments, the materials can be of any of the sizes described herein. In some embodiments, the encapsulated engineered cells are viable. In some embodiments, the encapsulated engineered cells are functional. In some embodiments, the engineered cells can be encapsulated in any of the photo- responsive materials described herein. In some embodiments, the engineered cells are encapsulated in sodium alginate microcapsules. In some embodiments, the engineered cells can be encapsulated in 0.4 mm capsules. In some embodiments, the engineered cells are encapsulated in 0.4 mm sodium alginate microcapsules.

[00236] In some embodiments, the cell engineered platform can include engineered cells that can be encapsulated into microcapsules (FIG. 7B). In some embodiments, the engineered cells can be encapsulated in any of the natural and/or synthetic materials described herein. In some embodiments, the therapeutic agent (e.g., pro-angiogenic or pro-lymphogenic factors) produced, expressed and/or secreted by the engineered cells encapsulated in the microcapsules can diffuse though the capsule and outside the capsule. In some embodiments, the engineered cells encapsulated in a microcapsule can secrete outside the capsule a therapeutic agent (e.g., pro- angiogenic or pro-lymphogenic factors), including, but not limited to, Epidermal Growth Factor (EGF), Platelet Derived Growth Factor A (PDGF-A), Vascular Endothelial Growth Factor A (VEGF-A), Vascular Endothelial Growth Factor C (VEGF-C), Placental Growth Factor (P1GF), and/or Fibroblast Growth Factors 2, 10 and 21 (FGF2, FGF10 and FGF21) (FIG. 7B). In some embodiments, the therapeutic agents secreted outside the capsule can guide/generate/induce the new lymphatic and blood vessels. [00237] In some embodiments, the cell engineered platform can include engineered cells that can be encapsulated into macro-platforms/capsules (macro-capsules) (FIG. 7C). In some embodiments, the macro-platform includes encapsulated engineered cells included into the bulk of any of the natural and/or synthetic materials described herein (FIG. 7C). In some embodiments, the bulk of the material can include a vascularizing channel. In some embodiments, the vascularizing channel can include an entry point. In some embodiments, blood and/or lymphatic vessels can enter the vascularizing channel through the entry point. In some embodiments, lymphatic and/or blood vessels can growth through the vascularizing channel. In some embodiments, the encapsulated engineered cells included in the bulk of the material can express, produce and/or secrete any of the therapeutic agents (e.g., pro-angiogenic or pro-lymphogenic factors) described herein, including, but not limited to, Epidermal Growth Factor (EGF), Platelet Derived Growth Factor A (PDGF-A), Vascular Endothelial Growth Factor A (VEGF-A), Vascular Endothelial Growth Factor C (VEGF-C), Placental Growth Factor (P1GF), and/or Fibroblast Growth Factors 2, 10 and 21 (FGF2, FGF10 and FGF21). In some embodiments, the lymphatic and/or blood vessels can enter the vascularized channel and/or growth inside the vascularized channel in response to the therapeutic agent (e.g., pro-angiogenic or pro-lymphogenic factors) secreted by the encapsulated engineered cells included in the bulk of the material (FIG. 7C). In some embodiments, the therapeutic agents can be secreted outside the capsule and can guide/generate/induce new lymphatic and blood vessels.

[00238] In some embodiments, the cell engineered platform can include engineered cells that can be perfused into a perfused platform (FIG. 7D). In some embodiments, the perfused platform can include any of the natural or synthetic materials described herein. In some embodiments, the bulk of the natural or synthetic the material can include a vascularizing perfused channel. In some embodiments, the engineered cells can be perfused into the vascularized perfused channel. In some embodiments, the vascularizing perfused channel includes perfused engineered cells that express, produce and/or secrete any of the therapeutic agents (e.g., pro-angiogenic or pro-lymphogenic factors) described herein including, but not limited to, Epidermal Growth Factor (EGF), Platelet Derived Growth Factor A (PDGF-A), Vascular Endothelial Growth Factor A (VEGF-A), Vascular Endothelial Growth Factor C (VEGF-C), Placental Growth Factor (P1GF), and/or Fibroblast Growth Factors 2, 10 and 21 (FGF2, FGF10 and FGF21). In some embodiments, the vascularizing perfused channel can include an entry point. In some embodiments, lymphatic and/or blood vessels can enter the vascularizing perfused channel through the entry point. In some embodiments, lymphatic and/or blood vessels can growth through the vascularizing perfused channel. In some embodiments, the lymphatic and/or blood vessels can enter the perfused vascularized channel and/or growth inside the perfused vascularized channel in response to the therapeutic agent (e.g., pro-angiogenic or pro-lymphogenic factors) secreted by the perfused engineered cells included in the vascularizing perfused channel (FIG. 7D). In some embodiments, the therapeutic agents secreted outside the capsule can guide/generate/induce the new lymphatic and blood vessels.

[00239] In some embodiments, any of the cell engineered platforms described herein including non-encapsulated clusters (FIG. 7A), micro-encapsulated engineered cells (FIG. 7B), macro-encapsulated engineered cells (FIG. 7C), perfused engineered cells (FIG. 7D), or combinations thereof, can be implanted into a subject. In some embodiments, the engineered cells can be implanted into a subject in vivo. In some embodiments the subject can be a human or a nonhuman mammal. Examples of non-human mammals include, but are not limited to, farm animals (e.g., cows, pigs, and horses), domesticated animals (e.g., dogs, cats, rabbits, and horses), human companion animals, zoo animals, wild animals, and laboratory animals (e.g., rats, mice, hamsters, guinea pigs, monkeys, and apes). In some embodiments, the subject is a SKH1 ELITE mice. In some embodiments, the subject is a pre-clinical rodent lymphedema model (hindlimb). In some embodiments, the subject is a human.

[00240] In some embodiments, the subject (e.g., lymphedema mouse model, human) undergoes radiation therapy and/or lymph node removal before implantation of any of the cell engineered platforms described herein. In some embodiments, the cell engineered platform includes an immunoprotective material that can limit, reduce and/or impede immune cells and/or radiation from entering the platform.

[00241] In some embodiments, the subject suffers from and/or is need of treatment for a disease or condition, including but not limited to, lymphedema, autoimmune disorders, metabolic diseases, oncologic related diseases, neurologic related diseases, pathologic inflammation, and cardiovascular disease.

[00242] In some embodiments, lymphedema in the subject can be assessed using magnetic resonance imaging (MRI) or any other suitable technique described in the art. In some embodiments, lymphedema in the subject can be assessed after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25, 30, 40, 50, 60, 70, or 90 days, 4, 5, 6, 7, 8, 9, 10, or 12 months, 1, 2, 3, 4, 5, 6, or 10 years from the day of implantation of any of the cell engineered platforms described herein. In some embodiments, lymphedema is assessed after 2 months from implantation of any of the engineered platforms described herein. In some embodiments, implantation of any of the cell engineered platforms described herein reduces lymphedema by about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%. In some embodiments, implantation in a mouse model of lymphedema of any of the cell engineered platforms described herein including engineered cells expressing VEGF-C can reduce lymphedema by about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%. In some embodiments, implantation in a mouse model of lymphedema of any of the cell engineered platforms described herein including engineered cells expressing VEGF-C reduces lymphedema by about 90% to about 99% (e.g., compared to an untreated control).

[00243] In some embodiments, the presence of a pro-angiogenic signal in a subject can be detected with IVIS imaging. In some embodiments, previous to IVIS imaging, a neovascularization probe can be administered to the subject. In some embodiments, the neovascularization probe can be injected into the subject. In some embodiments, the neovascularization probe is integrisense. In some embodiments, the engineered cells can be implanted in the subQ space of a mouse.

[00244] In some embodiments, any of the cell engineered platforms described herein including non-encapsulated clusters (FIG. 7A), micro-encapsulated engineered cells (FIG. 7B), macro-encapsulated engineered cells (FIG. 7C), perfused engineered cells (FIG. 7D), or combinations thereof, can be implanted into a subject using any surgical approach described in the art. In some embodiments, the platform can be implanted using a single and simple surgical approach. In some embodiments, the platform can be implanted using a non-invasive and/or short surgical approach. In some embodiments, the surgical approach removes the need for donor vessels and lymph nodes. In some embodiments, surgical implantation of any of the cell engineered platforms described herein can heal and/or regenerate lymphatic and/or blood vessels, and/or remove lymphedema. In some embodiments, surgical implantation of any of the cell engineered platforms described herein can heal and/or regenerate lymphatic and/or blood vessels, and/or remove lymphedema in greater than 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% of breast cancer related lymphedema (BCRL) patients. In some embodiments, the surgical approach includes an anti-fibrotic modification to avoid patient rejection. In some embodiments, the use of any of the cell engineered platforms described herein can eliminate mismatch donor vessels. In some embodiment, the platform can adjust the delivery of therapeutic agents based on the degree of edema. In some embodiments, the platform includes an implantable hydrogel platform engineered to guide and generate new lymphatic and blood vessels to the area of swelling to resolve chronic lymphedema.

[00245] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the molecular weight photo-responsive alginate is less than 95 kDa and wherein the methacrylation efficiency is from about 15% to about 95%; and at least one engineered cell.

[00246] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the molecular weight photo-responsive alginate is from about 55 kDa to about 240 kDa and wherein the methacrylation efficiency is from about 1% to about 65%; and at least one engineered cell.

[00247] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the molecular weight photo-responsive alginate is from about 180 kDa to about 320 kDa and wherein the methacrylation efficiency from about 1% to about 25%and at least one engineered cell.

[00248] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the molecular weight photo-responsive alginate is less than 95 kDa and wherein the methacrylation efficiency is from about 15% to about 95%; and at least one engineered cell, wherein the hydrogel system comprises a monomer of the formula (I):

[00249] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the molecular weight photo-responsive alginate is from about 55 kDa to about

240 kDa and wherein the methacrylation efficiency is from about 1% to about 65%; and at least one engineered cell, and wherein the hydrogel system comprises a monomer of the formula (I)

[00250] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the molecular weight photo-responsive alginate is from about 180 kDa to about 320 kDa and wherein the methacrylation efficiency from about 1% to about 25%; and at least one engineered cell, and wherein the hydrogel system comprises a monomer of the formula (I):

[00251] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel or lumen and at least one engineered cell.

[00252] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell.

[00253] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with blood cells, and at least one engineered cell.

[00254] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition further comprises polyethylene glycol diacrylate, gelatin methacrylate, a triazole-containing alginate, or any combination thereof, and at least one engineered cell.

[00255] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises gelatin methacrylate, and at least one engineered cell.

[00256] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises a triazole-containing alginate, and at least one engineered cell.

[00257] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate and at least one engineered cell, wherein the cell can express, produce and/or secrete a pro-angiogenic and/or a pro-lymphogenic factor.

[00258] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor A (VEGF-A).

[00259] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate and at least one engineered cell, wherein the cell can express, produce and/or secrete Placental Growth Factor (Pl GF).

[00260] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor C (VEGF-C).

[00261] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate and at least one engineered cell, wherein the cell can express, produce and/or secrete Epidermal Growth Factor (EGF).

[00262] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 2.

[00263] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 10.

[00264] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 21.

[00265] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel or lumen and at least one engineered cell, wherein the cell can express, produce and/or secrete Platelet Derived Growth Factor A (PDGF-A).

[00266] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel or lumen and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor A (VEGF-A).

[00267] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel or lumen and at least one engineered cell, wherein the cell can express, produce and/or secrete Placental Growth Factor (P1GF).

[00268] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel or lumen and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor C (VEGF-C).

[00269] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel or lumen and at least one engineered cell, wherein the cell can express, produce and/or secrete Epidermal Growth Factor (EGF).

[00270] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel or lumen and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 2.

[00271] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least one channel or lumen and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 10.

[00272] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive al ginate wherein the tissue mimicking composition comprises at least one channel or lumen and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 21.

[00273] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Platelet Derived Growth Factor A (PDGF-A).

[00274] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor A (VEGF-A).

[00275] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Placental Growth Factor (P1GF).

[00276] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor C (VEGF-C).

[00277] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Epidermal Growth Factor (EGF).

[00278] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 2.

[00279] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 10.

[00280] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 21.

[00281] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with blood cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Platelet Derived Growth Factor A (PDGF-A).

[00282] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor A (VEGF-A).

[00283] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Placental Growth Factor (P1GF).

[00284] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor C (VEGF-C).

[00285] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Epidermal Growth Factor (EGF).

[00286] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 2.

[00287] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 10.

[00288] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 21.

[00289] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition further comprises polyethylene glycol diacrylate, gelatin methacrylate, a triazole-containing alginate, or any combination thereof, and at least one engineered cell, wherein the cell can express, produce and/or secrete Platelet Derived Growth Factor A (PDGF-A).

[00290] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition further comprises polyethylene glycol diacrylate, gelatin methacrylate, a triazole-containing alginate, or any combination thereof, and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor A (VEGF-A).

[00291] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition further comprises polyethylene glycol diacrylate, gelatin methacrylate, a triazole-containing alginate, or any combination thereof, and at least one engineered cell, wherein the cell can express, produce and/or secrete Placental Growth Factor (P1GF).

[00292] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition further comprises polyethylene glycol diacrylate, gelatin methacrylate, a triazole-containing alginate, or any combination thereof, and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor C (VEGF-C).

[00293] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition further comprises polyethylene glycol diacrylate, gelatin methacrylate, a triazole-containing alginate, or any combination thereof, and at least one engineered cell, wherein the cell can express, produce and/or secrete Epidermal Growth Factor (EGF).

[00294] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition further comprises polyethylene glycol diacrylate, gelatin methacrylate, a triazole-containing alginate, or any combination thereof, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 2.

[00295] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition further comprises polyethylene glycol diacrylate, gelatin methacrylate, a triazole-containing alginate, or any combination thereof, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 10.

[00296] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition further comprises polyethylene glycol diacrylate, gelatin methacrylate, a triazole-containing alginate, or any combination thereof, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 21.

[00297] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least two channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Platelet Derived Growth Factor A (PDGF-A).

[00298] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least two channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor A (VEGF-A).

[00299] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least two channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Placental Growth Factor (P1GF).

[00300] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least two channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor C (VEGF-C).

[00301] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least two channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Epidermal Growth Factor (EGF).

[00302] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least two channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 2.

[00303] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least two channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 10.

[00304] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least two channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 21.

[00305] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein a boundary between adjacent channels of the tissue mimicking composition has one or more pores allowing contact between adjacent channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Platelet Derived Growth Factor A (PDGF-A). [00306] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein a boundary between adjacent channels of the tissue mimicking composition has one or more pores allowing contact between adjacent channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor A (VEGF-A).

[00307] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein a boundary between adjacent channels of the tissue mimicking composition has one or more pores allowing contact between adjacent channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Placental Growth Factor (P1GF).

[00308] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein a boundary between adjacent channels of the tissue mimicking composition has one or more pores allowing contact between adjacent channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor C (VEGF-C).

[00309] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein a boundary between adjacent channels of the tissue mimicking composition has one or more pores allowing contact between adjacent channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Epidermal Growth Factor (EGF).

[00310] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein a boundary between adjacent channels of the tissue mimicking composition has one or more pores allowing contact between adjacent channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 2.

[00311] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein a boundary between In some embodiments, the present disclosure provides adjacent channels of the tissue mimicking composition has one or more pores allowing contact between adjacent channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 10.

[00312] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein a boundary between adjacent channels of the tissue mimicking composition has one or more pores allowing contact between adjacent channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 21.

[00313] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition resembles a lymph node, and at least one engineered cell, wherein the cell can express, produce and/or secrete Platelet Derived Growth Factor A (PDGF-A).

[00314] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition resembles a lymph node, and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor A (VEGF-A).

[00315] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition resembles a lymph node, and at least one engineered cell, wherein the cell can express, produce and/or secrete Placental Growth Factor (P1GF).

[00316] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition resembles a lymph node, and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor C (VEGF-C).

[00317] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition resembles a lymph node, and at least one engineered cell, wherein the cell can express, produce and/or secrete Epidermal Growth Factor (EGF).

[00318] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition resembles a lymph node, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 2. [00319] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition resembles a lymph node, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 10. In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) in a subject (e.g., human, pig, mouse), wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition resembles a lymph node, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 21

[00320] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the molecular weight photo-responsive alginate is less than 95 kDa and wherein the methacrylation efficiency is from about 15% to about 95%; and at least one engineered cell.

[00321] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the molecular weight photo-responsive alginate is from about 55 kDa to about 240 kDa and wherein the methacrylation efficiency is from about 1% to about 65%; and at least one engineered cell.

[00322] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the molecular weight photo-responsive alginate is from about 180 kDa to about 320 kDa and wherein the methacrylation efficiency from about 1% to about 25%and at least one engineered cell.

[00323] In some embodiments, the present disclosure provides tissue mimicking compositions for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the molecular weight photo-responsive alginate is less than 95 kDa and wherein the methacrylation efficiency is from about 15% to about 95%; and at least one engineered cell, wherein the hydrogel system comprises a monomer of the formula (I):

[00324] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the molecular weight photo-responsive alginate is from about 55 kDa to about 240 kDa and wherein the methacrylation efficiency is from about 1% to about 65%; and at least one engineered cell, and wherein the hydrogel system comprises a monomer of the formula (I):

[00325] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the molecular weight photo-responsive alginate is from about 180 kDa to about 320 kDa and wherein the methacryl ati on efficiency from about 1% to about 25%and at least one engineered cell, and wherein the hydrogel system comprises a monomer of the formula (I):

[00326] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel or lumen and at least one engineered cell.

[00327] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell.

[00328] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with blood cells, and at least one engineered cell.

[00329] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition further comprises polyethylene glycol diacrylate, gelatin methacrylate, a triazole-containing alginate, or any combination thereof, and at least one engineered cell.

[00330] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises gelatin methacrylate, and at least one engineered cell.

[00331] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises a triazole-containing alginate.

[00332] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate and at least one engineered cell, wherein the cell can express, produce and/or secrete a pro-angiogenic and/or a pro-lymphogenic factor.

[00333] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate and at least one engineered cell, wherein the cell can express, produce and/or secrete Platelet Derived Growth Factor A (PDGF-A)

[00334] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor A (VEGF-A).

[00335] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate and at least one engineered cell, wherein the cell can express, produce and/or secrete Placental Growth Factor (Pl GF).

[00336] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor C (VEGF-C).

[00337] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate and at least one engineered cell, wherein the cell can express, produce and/or secrete Epidermal Growth Factor (EGF).

[00338] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 2.

[00339] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 10. [00340] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 21.

[00341] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel or lumen and at least one engineered cell, wherein the cell can express, produce and/or secrete Platelet Derived Growth Factor A (PDGF-A).

[00342] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel or lumen and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor A (VEGF-A).

[00343] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel or lumen and at least one engineered cell, wherein the cell can express, produce and/or secrete Placental Growth Factor (P1GF).

[00344] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel or lumen and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor C (VEGF-C).

[00345] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel or lumen and at least one engineered cell, wherein the cell can express, produce and/or secrete Epidermal Growth Factor (EGF).

[00346] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel or lumen and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 2. [00347] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least one channel or lumen and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 10.

[00348] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least one channel or lumen and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 21.

[00349] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Platelet Derived Growth Factor A (PDGF-A).

[00350] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor A (VEGF-A).

[00351] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Placental Growth Factor (P1GF).

[00352] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor C (VEGF-C).

[00353] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate, wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Epidermal Growth Factor (EGF).

[00354] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 2.

[00355] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 10.

[00356] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 21.

[00357] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with blood cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Platelet Derived Growth Factor A (PDGF-A).

[00358] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor A (VEGF-A).

[00359] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Placental Growth Factor (P1GF).

[00360] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor C (VEGF-C).

[00361] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Epidermal Growth Factor (EGF).

[00362] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 2.

[00363] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 10. [00364] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 21.

[00365] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition further comprises polyethylene glycol diacrylate, gelatin methacrylate, a triazole-containing alginate, or any combination thereof, and at least one engineered cell, wherein the cell can express, produce and/or secrete Platelet Derived Growth Factor A (PDGF-A).

[00366] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition further comprises polyethylene glycol diacrylate, gelatin methacrylate, a triazole-containing alginate, or any combination thereof, and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor A (VEGF-A).

[00367] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition further comprises polyethylene glycol diacrylate, gelatin methacrylate, a triazole-containing alginate, or any combination thereof, and at least one engineered cell, wherein the cell can express, produce and/or secrete Placental Growth Factor (Pl GF).

[00368] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition further comprises polyethylene glycol diacrylate, gelatin methacrylate, a triazole-containing alginate, or any combination thereof, and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor C (VEGF-C).

[00369] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition further comprises polyethylene glycol diacrylate, gelatin methacrylate, a triazole-containing alginate, or any combination thereof, and at least one engineered cell, wherein the cell can express, produce and/or secrete Epidermal Growth Factor (EGF).

[00370] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition further comprises polyethylene glycol diacrylate, gelatin methacrylate, a triazole-containing alginate, or any combination thereof, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 2.

[00371] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition further comprises polyethylene glycol diacrylate, gelatin methacrylate, a triazole-containing alginate, or any combination thereof, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 10.

[00372] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition further comprises polyethylene glycol diacrylate, gelatin methacrylate, a triazole-containing alginate, or any combination thereof, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 21.

[00373] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least two channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Platelet Derived Growth Factor A (PDGF-A).

[00374] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least two channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor A (VEGF-A).

[00375] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least two channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Placental Growth Factor (P1GF).

[00376] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least two channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor C (VEGF-C).

[00377] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least two channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Epidermal Growth Factor (EGF).

[00378] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least two channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 2.

[00379] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least two channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 10.

[00380] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition comprises at least two channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 21.

[00381] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein a boundary between adjacent channels of the tissue mimicking composition has one or more pores allowing contact between adjacent channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Platelet Derived Growth Factor A (PDGF-A). [00382] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein a boundary between adjacent channels of the tissue mimicking composition has one or more pores allowing contact between adjacent channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor A (VEGF-A).

[00383] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein a boundary between adjacent channels of the tissue mimicking composition has one or more pores allowing contact between adjacent channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Placental Growth Factor (P1GF).

[00384] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein a boundary between adjacent channels of the tissue mimicking composition has one or more pores allowing contact between adjacent channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor C (VEGF-C).

[00385] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein a boundary between adjacent channels of the tissue mimicking composition has one or more pores allowing contact between adjacent channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Epidermal Growth Factor (EGF).

[00386] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein a boundary between adjacent channels of the tissue mimicking composition has one or more pores allowing contact between adjacent channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 2.

[00387] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein a boundary between adjacent channels of the tissue mimicking composition has one or more pores allowing contact between adjacent channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 10.

[00388] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein a boundary between adjacent channels of the tissue mimicking composition has one or more pores allowing contact between adjacent channels, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 21.

[00389] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition resembles a lymph node, and at least one engineered cell, wherein the cell can express, produce and/or secrete Platelet Derived Growth Factor A (PDGF-A).

[00390] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition resembles a lymph node, and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor A (VEGF-A).

[00391] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition resembles a lymph node, and at least one engineered cell, wherein the cell can express, produce and/or secrete Placental Growth Factor (P1GF).

[00392] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition resembles a lymph node, and at least one engineered cell, wherein the cell can express, produce and/or secrete Vascular Endothelial Growth Factor C (VEGF-C).

[00393] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition resembles a lymph node, and at least one engineered cell, wherein the cell can express, produce and/or secrete Epidermal Growth Factor (EGF).

[00394] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition resembles a lymph node, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 2. [00395] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition resembles a lymph node, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 10. [00396] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of a disease or disorder (e.g., lymphedema) comprising obtaining at least one ink composition; and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition, optionally, wherein the method further comprises exposing the deposited ink pattern to light, wherein said tissue mimicking composition comprises a photo-responsive alginate wherein the tissue mimicking composition resembles a lymph node, and at least one engineered cell, wherein the cell can express, produce and/or secrete Fibroblast Growth Factor 21. [00397] In some embodiments, the present disclosure provides a tissue mimicking composition for use in the treatment of lymphedema in a human, wherein the composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 that can produce and secrete a pro-angiogenic and/or a pro-lymphogenic factor; optionally, wherein the engineered cells are encapsulated in alginate capsules.

[00398] In some embodiments, the present disclosure provides a tissue mimicking composition for use in the treatment of lymphedema in a human, wherein the composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 that can produce and secrete Platelet Derived Growth Factor A (PDGF-A); optionally, wherein the engineered cells are encapsulated in alginate capsules.

[00399] In some embodiments, the present disclosure provides a tissue mimicking composition for use in the treatment of lymphedema in a human, wherein the composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 that can produce and secrete Vascular Endothelial Growth Factor A (VEGF-A); optionally, wherein the engineered cells are encapsulated in alginate capsules.

[00400] In some embodiments, the present disclosure provides a tissue mimicking composition for use in the treatment of lymphedema in a human, wherein the composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 that can produce and secrete Placental Growth Factor (P1GF); optionally, wherein the engineered cells are encapsulated in alginate capsules.

[00401] In some embodiments, the present disclosure provides a tissue mimicking composition for use in the treatment of lymphedema in a human, wherein the composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 that can produce and secrete Vascular Endothelial Growth Factor C (VEGF-C); optionally, wherein the engineered cells are encapsulated in alginate capsules.

[00402] In some embodiments, the present disclosure provides a tissue mimicking composition for use in the treatment of lymphedema in a human, wherein the composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 that can produce and secrete Epidermal Growth Factor (EGF); optionally, wherein the engineered cells are encapsulated in alginate capsules.

[00403] In some embodiments, the present disclosure provides a tissue mimicking composition for use in the treatment of lymphedema in a human, wherein the composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 that can produce and secrete Fibroblast Growth Factor 2; optionally, wherein the engineered cells are encapsulated in alginate capsules.

[00404] In some embodiments, the present disclosure provides a tissue mimicking composition for use in the treatment of lymphedema in a human, wherein the composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 that can produce and secrete Fibroblast Growth Factor 10; optionally, wherein the engineered cells are encapsulated in alginate capsules.

[00405] In some embodiments, the present disclosure provides a tissue mimicking composition for use in the treatment of lymphedema in a human, wherein the composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 that can produce and secrete Fibroblast Growth Factor 21; optionally, wherein the engineered cells are encapsulated in alginate capsules.

[00406] In some embodiments, the present disclosure provides a tissue mimicking composition for use in the treatment of lymphedema in a mouse, wherein the composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 that can produce and secrete a pro-angiogenic and/or a pro-lymphogenic factor; optionally, wherein the engineered cells are encapsulated in alginate capsules.

[00407] In some embodiments, the present disclosure provides a tissue mimicking composition for use in the treatment of lymphedema in a mouse, wherein the composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 that can produce and secrete Platelet Derived Growth Factor A (PDGF-A); optionally, wherein the engineered cells are encapsulated in alginate capsules.

[00408] In some embodiments, the present disclosure provides a tissue mimicking composition for use in the treatment of lymphedema in a mouse, wherein the composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 that can produce and secrete Vascular Endothelial Growth Factor A (VEGF-A); optionally, wherein the engineered cells are encapsulated in alginate capsules.

[00409] In some embodiments, the present disclosure provides a tissue mimicking composition for use in the treatment of lymphedema in a mouse, wherein the composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 that can produce and secrete Placental Growth Factor (P1GF); optionally, wherein the engineered cells are encapsulated in alginate capsules.

[00410] In some embodiments, the present disclosure provides a tissue mimicking composition for use in the treatment of lymphedema in a mouse, wherein the composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 that can produce and secrete Vascular Endothelial Growth Factor C (VEGF-C); optionally, wherein the engineered cells are encapsulated in alginate capsules.

[00411] In some embodiments, the present disclosure provides a tissue mimicking composition for use in the treatment of lymphedema in a mouse, wherein the composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 that can produce and secrete Epidermal Growth Factor (EGF); optionally, wherein the engineered cells are encapsulated in alginate capsules.

[00412] In some embodiments, the present disclosure provides a tissue mimicking composition for use in the treatment of lymphedema in a mouse, wherein the composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 that can produce and secrete Fibroblast Growth Factor 2; optionally, wherein the engineered cells are encapsulated in alginate capsules.

[00413] In some embodiments, the present disclosure provides a tissue mimicking composition for use in the treatment of lymphedema in a mouse, wherein the composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 that can produce and secrete Fibroblast Growth Factor 10; optionally, wherein the engineered cells are encapsulated in alginate capsules.

[00414] In some embodiments, the present disclosure provides a tissue mimicking composition for use in the treatment of lymphedema in a mouse, wherein the composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 that can produce and secrete Fibroblast Growth Factor 21; optionally, wherein the engineered cells are encapsulated in alginate capsules.

[00415] In some embodiments, the present disclosure provides a tissue mimicking composition for use in the treatment of lymphedema in a pig, wherein the composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 that can produce and secrete Platelet Derived Growth Factor A (PDGF-A); optionally, wherein the engineered cells are encapsulated in alginate capsules.

[00416] In some embodiments, the present disclosure provides a tissue mimicking composition for use in the treatment of lymphedema in a pig, wherein the composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 that can produce and secrete a pro-angiogenic and/or a pro-lymphogenic factor; optionally, wherein the engineered cells are encapsulated in alginate capsules.

[00417] In some embodiments, the present disclosure provides a tissue mimicking composition for use in the treatment of lymphedema in a pig, wherein the composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 that can produce and secrete Vascular Endothelial Growth Factor A (VEGF-A); optionally, wherein the engineered cells are encapsulated in alginate capsules.

[00418] In some embodiments, the present disclosure provides a tissue mimicking composition for use in the treatment of lymphedema in a pig, wherein the composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 that can produce and secrete Placental Growth Factor (P1GF); optionally, wherein the engineered cells are encapsulated in alginate capsules.

[00419] In some embodiments, the present disclosure provides a tissue mimicking composition for use in the treatment of lymphedema in a pig, wherein the composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 that can produce and secrete Vascular Endothelial Growth Factor C (VEGF-C); optionally, wherein the engineered cells are encapsulated in alginate capsules.

[00420] In some embodiments, the present disclosure provides a tissue mimicking composition for use in the treatment of lymphedema in a pig, wherein the composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 that can produce and secrete Epidermal Growth Factor (EGF); optionally, wherein the engineered cells are encapsulated in alginate capsules.

[00421] In some embodiments, the present disclosure provides a tissue mimicking composition for use in the treatment of lymphedema in a pig, wherein the composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 that can produce and secrete Fibroblast Growth Factor 2; optionally, wherein the engineered cells are encapsulated in alginate capsules.

[00422] In some embodiments, the present disclosure provides a tissue mimicking composition for use in the treatment of lymphedema in a pig, wherein the composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 that can produce and secrete Fibroblast Growth Factor 10; optionally, wherein the engineered cells are encapsulated in alginate capsules.

[00423] In some embodiments, the present disclosure provides a tissue mimicking composition for use in the treatment of lymphedema in a pig, wherein the composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 that can produce and secrete Fibroblast Growth Factor 21; optionally, wherein the engineered cells are encapsulated in alginate capsules. In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of lymphedema in a human comprising obtaining at least one ink composition and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition; optionally, wherein the method further comprises exposing the deposited ink pattern to light; wherein said tissue mimicking composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 cells that produce and/or secrete Platelet Derived Growth Factor A (PDGF-A); optionally, wherein the cells are encapsulated in alginate capsules.

[00424] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of lymphedema in a human comprising obtaining at least one ink composition and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition; optionally, wherein the method further comprises exposing the deposited ink pattern to light; wherein said tissue mimicking composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 cells that produce and/or secrete Vascular Endothelial Growth Factor A (VEGF-A); optionally, wherein the cells are encapsulated in alginate capsules.

[00425] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of lymphedema in a human comprising obtaining at least one ink composition and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition; optionally, wherein the method further comprises exposing the deposited ink pattern to light; wherein said tissue mimicking composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 cells that produce and/or secrete Placental Growth Factor (P1GF); optionally, wherein the cells are encapsulated in alginate capsules.

[00426] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of lymphedema in a human comprising obtaining at least one ink composition and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition; optionally, wherein the method further comprises exposing the deposited ink pattern to light; wherein said tissue mimicking composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 cells that produce and/or secrete Vascular Endothelial Growth Factor C (VEGF-C); optionally, wherein the cells are encapsulated in alginate capsules.

[00427] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of lymphedema in a human comprising obtaining at least one ink composition and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition; optionally, wherein the method further comprises exposing the deposited ink pattern to light; wherein said tissue mimicking composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 cells that produce and/or secrete Epidermal Growth Factor (EGF); optionally, wherein the cells are encapsulated in alginate capsules.

[00428] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of lymphedema in a human comprising obtaining at least one ink composition and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition; optionally, wherein the method further comprises exposing the deposited ink pattern to light; wherein said tissue mimicking composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 cells that produce and/or secrete Fibroblast Growth Factor 2; optionally, wherein the cells are encapsulated in alginate capsules.

[00429] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of lymphedema in a human comprising obtaining at least one ink composition and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition; optionally, wherein the method further comprises exposing the deposited ink pattern to light; wherein said tissue mimicking composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 cells that produce and/or secrete Fibroblast Growth Factor 10; optionally, wherein the cells are encapsulated in alginate capsules.

[00430] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of lymphedema in a human comprising obtaining at least one ink composition and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition; optionally, wherein the method further comprises exposing the deposited ink pattern to light; wherein said tissue mimicking composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 cells that produce and/or secrete Fibroblast Growth Factor 21; optionally, wherein the cells are encapsulated in alginate capsules.

[00431] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of lymphedema in a mouse comprising obtaining at least one ink composition and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition; optionally, wherein the method further comprises exposing the deposited ink pattern to light; wherein said tissue mimicking composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 cells that produce and/or secrete Platelet Derived Growth Factor A (PDGF-A); optionally, wherein the cells are encapsulated in alginate capsules.

[00432] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of lymphedema in a mouse comprising obtaining at least one ink composition and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition; optionally, wherein the method further comprises exposing the deposited ink pattern to light; wherein said tissue mimicking composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 cells that produce and/or secrete Vascular Endothelial Growth Factor A (VEGF-A); optionally, wherein the cells are encapsulated in alginate capsules.

[00433] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of lymphedema in a mouse comprising obtaining at least one ink composition and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition; optionally, wherein the method further comprises exposing the deposited ink pattern to light; wherein said tissue mimicking composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 cells that produce and/or secrete Placental Growth Factor (P1GF); optionally, wherein the cells are encapsulated in alginate capsules.

[00434] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of lymphedema in a mouse comprising obtaining at least one ink composition and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition; optionally, wherein the method further comprises exposing the deposited ink pattern to light; wherein said tissue mimicking composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 cells that produce and/or secrete Vascular Endothelial Growth Factor C (VEGF-C); optionally, wherein the cells are encapsulated in alginate capsules.

[00435] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of lymphedema in a mouse comprising obtaining at least one ink composition and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition; optionally, wherein the method further comprises exposing the deposited ink pattern to light; wherein said tissue mimicking composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 cells that produce and/or secrete Epidermal Growth Factor (EGF); optionally, wherein the cells are encapsulated in alginate capsules.

[00436] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of lymphedema in a mouse comprising obtaining at least one ink composition and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition; optionally, wherein the method further comprises exposing the deposited ink pattern to light; wherein said tissue mimicking composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 cells that produce and/or secrete Fibroblast Growth Factor 2; optionally, wherein the cells are encapsulated in alginate capsules. [00437] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of lymphedema in a mouse comprising obtaining at least one ink composition and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition; optionally, wherein the method further comprises exposing the deposited ink pattern to light; wherein said tissue mimicking composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 cells that produce and/or secrete Fibroblast Growth Factor 10; optionally, wherein the cells are encapsulated in alginate capsules.

[00438] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of lymphedema in a mouse comprising obtaining at least one ink composition and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition; optionally, wherein the method further comprises exposing the deposited ink pattern to light; wherein said tissue mimicking composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 cells that produce and/or secrete Fibroblast Growth Factor 21; optionally, wherein the cells are encapsulated in alginate capsules.

[00439] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of lymphedema in a pig comprising obtaining at least one ink composition and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition; optionally, wherein the method further comprises exposing the deposited ink pattern to light; wherein said tissue mimicking composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 cells that produce and/or secrete Platelet Derived Growth Factor A (PDGF-A); optionally, wherein the cells are encapsulated in alginate capsules.

[00440] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of lymphedema in a pig comprising obtaining at least one ink composition and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition; optionally, wherein the method further comprises exposing the deposited ink pattern to light; wherein said tissue mimicking composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 cells that produce and/or secrete Vascular Endothelial Growth Factor A (VEGF-A); optionally, wherein the cells are encapsulated in alginate capsules.

[00441] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of lymphedema in a pig comprising obtaining at least one ink composition and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition; optionally, wherein the method further comprises exposing the deposited ink pattern to light; wherein said tissue mimicking composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 cells that produce and/or secrete Placental Growth Factor (P1GF); optionally, wherein the cells are encapsulated in alginate capsules.

[00442] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of lymphedema in a pig comprising obtaining at least one ink composition and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition; optionally, wherein the method further comprises exposing the deposited ink pattern to light; wherein said tissue mimicking composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 cells that produce and/or secrete Vascular Endothelial Growth Factor C (VEGF-C); optionally, wherein the cells are encapsulated in alginate capsules.

[00443] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of lymphedema in a pig comprising obtaining at least one ink composition and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition; optionally, wherein the method further comprises exposing the deposited ink pattern to light; wherein said tissue mimicking composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 cells that produce and/or secrete Epidermal Growth Factor (EGF); optionally, wherein the cells are encapsulated in alginate capsules.

[00444] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of lymphedema in a pig comprising obtaining at least one ink composition and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition; optionally, wherein the method further comprises exposing the deposited ink pattern to light; wherein said tissue mimicking composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 cells that produce and/or secrete Fibroblast Growth Factor 2; optionally, wherein the cells are encapsulated in alginate capsules.

[00445] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of lymphedema in a pig comprising obtaining at least one ink composition and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition; optionally, wherein the method further comprises exposing the deposited ink pattern to light; wherein said tissue mimicking composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 cells that produce and/or secrete Fibroblast Growth Factor 10; optionally, wherein the cells are encapsulated in alginate capsules.

[00446] In some embodiments, the present disclosure provides a method of making a tissue mimicking composition for use in the treatment of lymphedema in a pig comprising obtaining at least one ink composition and depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology to form a tissue mimicking composition; optionally, wherein the method further comprises exposing the deposited ink pattern to light; wherein said tissue mimicking composition comprises a photo-responsive alginate and a plurality of engineered ARPE-19 cells that produce and/or secrete Fibroblast Growth Factor 21; optionally, wherein the cells are encapsulated in alginate capsules.

[00447] Other Embodiments

[00448] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[00449] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[00450] Examples

To facilitate a better understanding of the present disclosure, the following examples of specific embodiments are given. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure. In no way should the following examples be read to limit or define the entire scope of the disclosure.

[00451] Provided herein, in certain embodiments, are tissue mimicking compositions which, in some embodiments, comprise one or more vessel mimicking channels. The present invention contemplates a variety of arrangements and relationships between the vessel mimicking channels of the presently disclosed compositions (see, for example, FIG. 1, FIG. 2, FIG. 3). The vessel-mimicking channels are surrounded by a semi-permeable biomaterial, which in some embodiments comprises a therapeutic agent. In some embodiments, the semi-permeable biomaterial is perforated or comprises pores which allow the transfer of material between vessel mimicking channels. Details of the presently disclosed compositions are provided below.

Example 1 - 3D bioprinted lymphatic tissue graft models

[00452J Open and perfusable hydrogel channels were 3D printed using light-based photoprojection printing. A bioink composed of 10wt% gelatin methacrylate (GelMA) and 3.25% polyethylene glycol diacrylate (PEGDA) was printed at 6 second exposure time at a 38% light intensity. Hydrogel architectures are designed and exported as STL files for printing. A single channel lymphatic mimic was printed and a dual channel lymphatic vessel and blood vessel mimics were printed that contained either one or two blood vessel adjacent to the lymphatic vessel. A 3D printed lymph node model was also printed with blood vessels that were superimposed on lymphatic vessels. All channels were perfused with ink to ensure perfusability of 3D printed structures. Renderings and printed hydrogel compositions are shown in FIG. 4.

Example 2 - Perfusion staining of lymphatic endothelial cells within 3D printed grafts

[00453] Lymphatic endothelial cells (LEC) were perfused into open channel 3D printed hydrogels from 5xl0⁶ - 30xl0⁶ cells/ml. Hydrogels were rotated for 2-4 hours after initial perfusion and then placed into a perfusion cassette for sequential daily perfusions and are connected to a perfusion pump with flowrates from 0.5-5uL/min. Hydrogels were imaged daily to monitor cell adhesion and coverage (FIG. 5). LECs were then fluorescence stained for LEC (green) and nuclei (DAPI) to evaluate channel coverage post-perfusion (FIG. 6).

Example 3 - Pro-lymphangiogenic and pro-angiogenic cell engineering platform

[00454J Described herein are compositions of matter for a pro-lymphangiogenic and pro- angiogenic cell engineered platform.

[00455] Embodiments as described herein can be used for long-term disease management, drug delivery platform, engineered cell therapy, vascular, vascularizing materials/therapy, and/or regenerative material/therapy.

[00456] Other uses for this platform include, but are not limited to, production of lymphatic and blood regenerative molecules and regeneration/growth/maintenance/maturation of lymphatic and blood vessels for but not limited to therapeutics for disease management. For example, embodiments can comprise vascularized hydrogel platforms that include but are not limited to tissue grafts and implants for the treatment of various diseases such as autoimmune disorders, metabolic diseases, oncology, neurology, inflammation, cardiovascular disease, and others.

[00457] The vascularizing engineered platforms described herein comprise desired parameters that can be tuned for production, potency, release of therapeutics.

[00458] Described herein is a cell engineering platform for lymphangiogenesis and angiogenesis. The cell engineering platform allows but is not limited to the controlled and uncontrolled secretion of pro-angiogenic and pro-lymphogenic growth factors. This cell engineering platform is based on engineering cells (e.g., ARPE-19 retinal pigment epithelial cells) using a piggyback transpose system for the insertion of a selected DNA fragment.

[00459] The engineered cells described herein can be encapsulated in synthetic and/or natural materials. The engineered platform can be used for promoting lymphatic regeneration, vascular regeneration, guiding lymphatic growth, and guiding vascular growth.

[00460] The cell engineering platform described herein can be used, but is not limited to, therapeutic applications and disease management. The cell engineering platform can secrete pro- angiogenic growth factors, including but not limited to Epidermal Growth Factor (EGF), Platelet Derived Growth Factor A (PDGF-A), Vascular Endothelial Growth Factor A (VEGF-A), Placental Growth Factor (P1GF), Fibroblast Growth Factor 2, 10 and 21 (FGF2, FGF10 and FGF21) in a controlled or uncontrolled dosage. [00461] The cell engineering platform described herein can secrete pro-lymphogenic growth factors, including, but not limited to, Vascular Endothelial Growth Factor C (VEGF-C) in a controlled dosage.

[00462] The cell engineering platforms described herein can be modified to secrete pro- angiogenic or pro-lymphogenic factors across different species.

[00463] The engineered cells can be modified to incorporate various quantities of DNA to modulate secretion of the pro-lymphogenic or pro-angiogenic protein.

[00464] The cell engineering method described herein can be modulated to use different promoters, linkers, secretion tags, signaling tags to modify secretion of desired growth factors.

[00465] Secretion of proteins from the engineered cells described herein can be enhanced by creating a monoclonal cell line that increases the production of a selected protein, and/or stabilizes production of selected protein during expansion of the cell line.

[00466] The engineered cells described herein can be encapsulated in natural and synthetic materials of different sizes while maintaining high levels of viability and functionality. The engineered cells can be encapsulated in microcapsules (FIG. 7B), in macro-platforms (FIG. 7C), photo-responsive materials, and the like.

[00467] The engineered cells described herein can be implanted in vivo via injection of engineered cells with or without an encapsulation platform.

[00468] The engineered cells described herein can be perfused into open channels of a natural or synthetic material platform (FIG. 7D), encapsulated in the bulk of natural or synthetic material (FIG. 7C), and/or infused into the area of interest with or without an encapsulation material (FIG. 7 A).

Example 4 -Cell engineered platform for lymphedema

[00469] Described herein is a platform that can be used for resolving medical conditions and diseases including, but not limited to, lymphedema. The platform includes a living regenerative factory, an immune protective material, and a regenerative guiding hydrogel. The method includes a single and simple surgical approach that can remove the need for donor vessels and lymph nodes (FIG. 18).

[00470] The living regenerative factory includes cells (e.g., ARPE-19 cells) engineered to produce and secrete a therapeutic molecule (e.g., VEGF-C). The regenerative guiding hydrogel includes open channels that can recruit blood and lymphatic vessels. The immune protective material includes an immune protective molecule modification that does not allow penetration of radiation into the hydrogel and/or penetration of cells from the immune system (FIG. 19).

[00471] This method includes an implantable hydrogel platform engineered to guide and generate new (de novo) lymphatic and blood vessels to the area of swelling to resolve chronic lymphedema. This platform can be adjusted to eliminate mismatch donor vessels. The method includes an anti-fibrotic modification to avoid patient rejection. This method can allow the incorporation of therapeutic agents that can be adjusted based on the degree of edema (FIG. 18). As shown in FIG. 20, implantation of the platform composition described in FIG. 19 in the lymph nodes resected from a lymphedema mouse model that undergo radiation therapy shows a significant reduction of limb swelling after 2 months when compared to the control.

Example 5: Generation and screening of alginate methacrylate (AIMA) formulation library yields stable hydrogels for RPE-VEGFC cell factory encapsulation

[00472] The incorporation of methacrylate groups in the alginate backbone enables them to rapidly crosslink in response to UV light and enhance their strength and stability. We synthesized alginate methacrylates (AIMA) in-house by reacting sodium alginates of very low viscosity (VLVG) with molecular weight < 75 kDa and low viscosity (LVG-20) also known as SLG20 with molecular weight 75 kDa - 220 kDa, at methacrylation efficiencies between 20-46% with 2- aminoethyl methacrylate (AEMA). We previously characterized AIMA using the 1H NMR. The characteristic peaks for the methylene protons in the region 5.5 - 6.2 ppm confirmed the methacrylation.

[00473] We examined the ability of AIMA to undergo gelation and form hydrogels that could retain the shape of their mold upon exposure to UV light. Gelation and hydrogel shape retention were evaluated for the ability of the AIMA formulation to take the shape of a 96-well plate. The hydrogels that retained the shape of mold were further screened for mechanical strength. To test for mechanical strength, VLVG and SLG20 AIMA were added into 96 well plates at lOOuL per well and placed under UV exposure at 4mW/cm^A2 for 15 minutes. These AIMA gels were retrieved from the 96-well plate and used for compression testing. Compression testing was performed by applying a constant crosshead linear rate of 10 um/s. Compression testing was performed until hydrogel fracture. Modulus was measured from the slope of stress vs. strain, limited to the fracture point. We were able to identify leading A1MA hydrogel formulations (VLVG-M46 and LV20-M20) as novel strong alginates that will help to maintain the stability of transplanted cells and therapeutics.

[00474] Methods

[00475] Engineering and culture of RPE-VEGFC cellular therapy with safety switch [00476] RPE-19 cells were engineered to secrete VEGF-C growth factor via a PiggyBac vector system with Lipofectamine 3000 (ThermoFisher, #L3000001) to transfect DNA inside RPE cells.

[00477] Alginate methacrylate (A1MA) synthesis and library generation

[00478] Alginate methacrylate (A1MA) was synthesized by reacting sodium alginates of different molecular weights (UPVLVG with molecular weight < 75 kDa, SLG-20 with molecular weight 75 kDa - 220 kDa and SLG-100 with molecular weight 200 kDa-300 kDa) with 2- aminoethyl methacrylate (AEMA) for 24 h. The product was dialyzed against DI water for 3 days, filtered through 70 micron pore sized filters and freeze dried. Alginates were stored at -20 degree Celsius until further use. Alginate methacrylate was then characterized using the 1H NMR. The characteristic peaks for the methylene protons in the region 5.5 - 6.2 ppm confirmed the methacrylation.

Table 3

ME: Methacrylation efficiency, M: Methacryl ati on

[00479] Rhodamine B gelation assay in vitro

[00480] A1MA formulations are added into 96 well plates at lOOuL with luL of lw/v% Rhodamine B in DMSO and then placed under UV light at 4rnW/cm^A2 for 15minutes. The plates are incubated for 10 minutes and then washed three times with DI water. Fluorescence measurements were taken (540nm excitation, 580nm emission) followed by gross imaging to access shape retention of the hydrogels within the 96 well plate. The negative control for the study was DI water and our positive control was a previously published alginate composed from 1.4wt/v% SL20 ionically crosslinked in barium chloride solution for 30 minutes. Gelation was determined by the fluorescence intensity averaging above 15,000 AUC.

[00481] Mechanical strength

[00482] A1MA formulations were added into 96 well plates at lOOuL per well and placed under UV exposure at 4mW/cm^A2 for 15 minutes. These A1MA gels were retrieved from the 96 well plate and used for compression testing. Compression testing will be performed by applying constant crosshead speed 1.0 cm/min and 10N. Compression test will be performed until fracture. Stress vs strain and modulus will be collected from the compression testing. Modulus will be obtained from the slope of stress vs strain, limited to the first 2% of strain.

[00483] RPE cell viability encapsulated within A1MA

[00484] ARPE-19 cells were seeded into black 96 well plates at 10k cells/90uL and given 24 hours to adhere and form monolayers. A1MA formulations were added into 96 well at 20uL per well along with 60uL of cell culture media. Alamar blue was then added into the wells at lOuL per well and plates were incubator at 37C for 3 hours. Fluorescence readings using TECAN plate reader (570 nm for measurement wavelength and 600 nm for reference wavelength). ARPE-19 ells in phenol red-free media alone were used as the positive control.

[00485] Table 4. Complete A1MA formulation library.

[00486] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

REFERENCES

[00487] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

Claims

WHAT IS CLAIMED IS

1. A method for treating a disease or disorder in a patient in need thereof, said method comprising embedding a tissue mimicking composition in the patient, wherein said tissue mimicking composition comprises:

(ii) at least one therapeutic agent.

The method of claim 1, wherein the hydrogel system comprises a monomer of the formula:

wherein n and m are each independently at least 1.

3. The method according to either claim 1 or claim 2, wherein the tissue mimicking composition comprises at least one channel or lumen.

4. The method according to any one of claims 1-3, wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with lymphatic cells.

5. The method according to any one of claims 1-4, wherein the tissue mimicking composition comprises at least one channel suitable for perfusion with blood cells.

6. The method according to any one of claims 1-5, wherein the tissue mimicking composition further comprises polyethylene glycol diacrylate, gelatin methacrylate, a triazole-containing alginate, or any combination thereof.

7. The method according to any one of claims 1-6, wherein the tissue mimicking composition comprises gelatin methacrylate.

8. The method according to any one of claims 1-7, wherein the tissue mimicking composition comprises a triazole-containing alginate.

9. The method according to any one of claims 1-8, wherein at least one therapeutic agent is an engineered cell or a non-engineered cell.

10. The method according to any one of claims 1-9, wherein at least one therapeutic agent is an engineered protein, a secreted protein, a hormone, a cytokine, an antibody, an enzyme, or a peptide.

11. The method according to any one of claims 1-10, wherein at least one therapeutic agent is a small molecule.

12. The method according to any one of claims 1-11, wherein at least one therapeutic agent is an agent used to treat or prevent inflammation.

13. The method according to any one of claims 1-12, wherein the tissue mimicking composition comprises at least two channels.

14. The method according to claim 13, wherein a boundary between adjacent channels of the tissue mimicking composition has one or more pores allowing contact between adjacent channels.

15. The method according to any one of claims 1-14, wherein the tissue mimicking composition resembles a lymph node.

16. The method according to any one of claims 1-15, wherein the disease or disorder is lymphedema or another lymphatic disease or disorder, an autoimmune disease or disorder, a metabolic disease or disorder, cancer, a neurological disease or disorder, inflammation, or cardiovascular disease.

17. The method according to any one of claims 1-16, wherein the disease or disorder is lymphedema or another lymphatic disease or disorder.

18. The method according to any one of claims 1-17, wherein the disease or disorder is lymphedema.

19. A method of making a tissue mimicking composition for use according to any one of claims 1-18 comprising: a) obtaining at least one ink composition; and b) depositing the obtained ink composition or compositions according to a pattern using an additive manufacturing technology; to form a tissue mimicking composition.

20. The method of claim 19, wherein the method further comprises exposing the deposited ink pattern to light.